HU229968B1 - Streptococcus vaccine - Google Patents

Abstract

The present invention relates to the field of bacterial polysaccharide antigen vaccines. In particular, the present invention relates to vaccines comprising a pneumococcal polysaccharide antigen, typically a pneumococcal polysaccharide conjugate antigen, formulated with a protein antigen form Streptococcus pneumoniae, and optionally a Th1-inducing adjuvant.

Description

BACKGROUND OF THE INVENTION Bacterial polysaccharide antigen vaccines. manufacture of digs Do you like that? pobssaebridges for use in therapeutic agents.

More specifically, the present invention relates to three related embodiments. --- corticosterized phthalocytes, antispasmodic pnesistocotitis: conjugated polysaccharide anugene-containing vaccines, formulated with Streptococcus xanthene antigen and optionally with Th1-inducing adjuvant; < - > specific preferred pneamococcal conjugates formulated with Th1 adjuvant; and 3 microbial polysaccharide conjugates generally conjugated to H. influenza D protein.

Sfeepfilieceejis pnaawonme is a Gram-positive bocephalus that is responsible for juvenile morbidity and mortality (especially in the young and the elderly) and includes invasive diseases such as pneumonia, bacteremia and meningitis, e.g. The proportion of pneumococcal pneumonia in the United States of America among people over 60 is about 3 to about 10000 people. In about 2% of cases, this leads to bafeteremia and other manifestations such as corneal throat and mortality. ; even with antibiotic treatment.

The pseudo-bacterial dye is encapsulated by a chemically linked polysaccharide which results in scrotypic differentiation and has 90 known pneumococcal sorotypes. and the envelope is a fundamental compelling factor for phteamicococci: viral healing, whereas barons not only protect the feactenitb: its internal surfaces from complement, the heme itself is scve weak immunogen o However, they can stimulate the immune system through a cross-linking mechanism by cross-linking B-ssj surface receptors.

Numerous experiments have shown that, due to invasive psaumeccus disease, protection fegjobiw correlates with the amount of specific antibodies in the feudal lobe and: protection is serotype-specific,

Vaccines based on polysaccharide-a-tigene are well known in the art. Also, the four human alci35 <3 ^ AJS. This mediated relationship includes the polysaccharide Vs polysephearid of Jöpato, the PRP polis & charya of Jitato / JMtö ·, T <iííVf? Zue. fetal acyclic vaccine containing A, C, W135 and osp serospections, and 23-valent pneumococcal vaccine, which is 1, 2, 3, 4, 5, 6B, 7F, S, 9N, 9V, 10A, 1A, 12F. , 14, 15B, I7F, 1SC, I9A, 19F, 20, 22F, 23F and 33 serotype types are still polysehydride (which contains at least 98% of the blood-derived pnm-nocturns).

The latter three vaccines provide protection against bacterial pathogens causing respiratory infections, which result in severe .morbidity and mortality in infants, but ezsro stitches have not been authorized for use in children under 2 years of age and are not sufficiently immunogenic in this age group. Esgi. Med. 310,1361-1566, p. (1934) j. The «Tepirociecas · pííá mo« w is invasive bacterial disease and middle-aged disease; the most common cause among infants and young children. Elderly people also develop a poor response to pseudococcal vaccines [Rögbmann et al., J. Gerontol 42, 265-2, p. t? 987) J, here is the bacterial lung Act the s z amu r. k: 9 52 2 7 - 3 0? Proliferation of 2A / LT ζ-inflammation in this population [Verghese and Berk, Medlaine (Saliteeres) 62,271-285]. p. (I9S3)}.

Λ Lack of immunogenicity in infants. Strategies designed to combat this include linking polytocharids to large immunomodulatory proteins, which provide adjunct T-cell help, and which elicit immunological memory against the polysaccharide ajxtigens to which they are co-engineered. Pnetnococcal conjugated gllkoprotem vaccine designs would still be tested for safety, mmwoogemlss, and bathtub performance.

The following relates to embodiment 1, which contains pseiwmkokfatsz polysaccharides.

The 23-valence rope-jugular pulmonococcal vaccine vein shows a wide variation in clinical efficacy up to 8% w / w _; [Fedson et al., Arch. Intern. Med- · 154, 2531-2535. p. (1994)}, Efficacy with Immunized Risk Groups, such as the Elderly, Bodgkm's Disease, Spleen Spleen Disease, and Sickle Cell Disease, and ognmntaglobulmaemias, et al., Arcán Intern. Med., 154, 2666-2677. (1994)}, and the disease tnanifesttae (iva Is. The 23-valent vaccine does not protect against psm: m ?, occult pneumonia (some seedy risk esoposfokbtu :, mim, e.g., Elderly) and otitis media.

Thus, there is a need for improved ptmnmolcolins vaccine formulations, preferably those that are more generous in preventing and ameliorating pnoamouscritic diseases (colds) in the elderly and infants.

The following is a list of 2, »exfoliated surface mock coconut polybob binder jugs and 3D-Ws; containing an implementation code.

It is generally accepted that commercially available ptmumofeekfcos-oo-oo-oagic acid is generally or slightly dependent on the concentration of plenar material induced by blending; In particular, polysaccharide 23 was licensed solely on the basis of the tamaeogenesis of arcs component components (Williams et al., Msw York Aeaccmy of Seienses, 241-249 (1995)). who react with a protective antibody to the first vaccine of the pofesaccharide or polysaccharide conjugate, and may reduce the dose and the number of vaccinations required to elicit protective immunity against infections caused by .SSsre ^ zococm zmesnwfo.

2S. Since the beginning of the 20th century, a large number of catatypes have been experimenting with compounds that, when added to antigens, improve their immunogenesis in ohoasyng hands (Summary: Powell and Newmafc »" Vseeine Design - the Snhunrt. aaá Adjávant Ápproaoh ", Chapter 7," Á compendioru " xcipients ^<!, published by Plenaro Press NY '(1995)} Many very potent but significant local and systemic adverse reactions are excluded, which preclude their use in human dermatological preparations. Aluminum-based adjuvants (as in the case of ⁵ alum. kidroxjd, alpha-phosphorus phosphorus, first described by 192 fean, is an exclusive immunological adruvans of US approved human vaccine.

Examples of alumíniutn based adjuvants adjovánsok the carrier class of artificial stone which are triggered by the "depot effect · * ^5th The antigen adsorbs to the surface of the adjuvant and, when the composition is injected, the adjuvant and? immediate delivery into the blood stream, but the formulation remains in the local injection environment and results in a more pronounced immune response. Another known advantage of such carrier adjuvanted hexafen is its ability to stabilize degradation-sensitive antigens, such as polysaccharide antigens.

30-MPL is an example of a non-carrier sdtnWtsnk. Complete; its name is-o-desaedos-mosofoslbril Spid & (or 3-De-O-acylmophosphoryl lipid A or 3- <Xdezae3k4Mn «ofisbrtl lipid Aj) and is called SD-MFL because it expresses that 3-position de-O-acetylated at the reducing end, de-O-acetylated as disclosed in GB 2220211. A mixture of S-deacylmopophosphoryl lipid A chemically 4-, 5- or o-allyl. It was prepared in the early 1990s, when the procedure for the 3-O-doubling of the snow derivative of 4'monotasefotsl resulted in a molecule that was further weakened without altering its immunostimulatory activity.

The 3D-MPL-; or alone, or preferably in combination with depot carrier adjuvants, such as almnimttm-httfeoxide, ammonium phosphate or oil-in-water emulsions. In such formulations, 3I &lt; RTI ID = 0.0 > MPL &lt; / RTI &gt; and aaftgene can be found in the same cassette, which allows for more efficient targeting of antigen and immune stimuli. Studies have shown that 3DMPL can further potentiate the antimicrobial Immune; [Thoelert et al., Vaeeine juice, 708-714. p. (199F>; BP §89454 Bj) Such combinations are advantageous in the literature for adsorption-prone (for example, bacterial polysaccharide conjugate antigen), since adsorption to the atom is advantageous for the stagnation of the substituent. Bvfek, in particular, are currently approved for use in human killing and vaccines, and vaccines containing SÖ MFt in combination with apple and alpha stdjuyantsss are preferred in the literature because of their ease of development and speed of market introduction.

Non-3-deaclial has been investigated in MFLA by adjuvant sputum with a number of monovalent phobsaeharide kst-ugatunian substance antigen. Co-injection of MPL in saline enhanced the serum eHealth response to four motto-valent polysaccharide conjugates: pneumococcal FSBB tetanusAoxoid, pneumococcal FS1.2d.differentia toxoid, and S.sw.sw.sf.w. . marked <* otevnu.hoz komugálva ÍSctmeenmn c »míst.t .J Immunolögv 54 ?, Ί> 6-2140 o'd {l ^Ci 9Ítt encapsulated responses were gotsdoiiák, mountain-antigés spéciSms WL oil -vls« moháéban ( carrier type (adjuvant) consistently enhanced the salt effect of MPL due to the fact that WL and antigen were present in the same particle, and by other polysulphide canaryngine; even in the case of vaccines.

Devi et al. [infect, immun. 59, 3790-3707. p. <1991) investigated the adjuvant effect of non-3-deacetylated MFL on salt anti-murine antibody against Qypioeoeeus btaofepolysaccharide. When MFL was co-administered with co-oligomers, there was only a slight increase in Ig-M and IgG-specific response to polysaccharide, but a much greater effect on MPL; was when the practicality of using the bs 4 / o'an immunization salt was given 2 days after the conjugate, which calls for a delay in our MPL bcad compared to the antigen, especially in infants. The adjuvant effect of MPL on the polysaccharides and polysaccharide fatty acids in ready-to-use Moeye-Nggonck seems to be sustained by the inclusion of MPL-nck in a suitable low-throughput delivery system. provides an effect and bypasses the problem of timing and delayed administration

In summary, the state of the art teaches that certain polysaccharide or polysaccharide co-antigens, wherein MPL or 3D-MPL are combined with murine, preferably alpha

Surprisingly, it has been found that, in certain poeomococcal pertussis hemorrhagic lesions, the vaccine-ready protein has a significantly higher self-mutation rate when the antigen is formulated with 3D-MPL1 alone and with adjuvant 3D-MPL, for example, aluminum-based. In addition, snegiigtell Java is independent of: the machine monomerization of the 3D-MPL used and whether the conjugate is present in a tnosovalent composition or combined to form a polyvalent composition.

The following relates to Embodiment 3 containing conjugate elements of bacterial bacillar protein D.

As mentioned above, acaciafesrid-based silicon-based vaccines are well known in the art. The clinical efficacy of the aforementioned authorized pttSaobartate vaccines is variable. The efficacy of the V-polysaccharide killing agent is estimated to be 55% to 77% for the prevention of cultured typhoid [Plotkla and Cam, Áre. Intern. Med 155,2293-2299. oki, {1995}], 79 The efficacy of the C-polysaccharide vaccine for menmimecoccus is 79% under epidemic conditions [Öe Ws et al., Ball, Work! Health Orgart, 74, 407-411. 23, (1996) The 23-valent pneumococcal vaccine showed a broad range of clinical efficacy, ranging from 0% to S1% (Fedssn et al., Arab. Bel. Mecl. i59, 2531-2555, 1994). )] As added up, it is accepted that the effectiveness of the pneumococcal ointment depends more or less on the effect of the e &gt;

In the face of immunization with a face-to-face approach to immunization, the problem is that polysaccharides themselves are weak in immunity. Strategies designed to overcome this deficiency of hrt immunogenicity include the linkage of polysaccharides to large rmtmogenic proteins, which translocate to provide T-cell help,

Examples of these highly common carriers currently commonly used include diphtheria toxoid (DT or CKM197 mastic), hypothalamus (TT), hemorrhoids (keybols bmpet) (K.LH), and purified fatty tissue from taberkohn.

Ace relating to commonly used carriers; problems

All of these commonly used carriers are associated with a number of problems, including the production of cpsjig inhibitors GM1 * and conjugate tetanus immunoglobulin signaling,

Despite their widespread use in the delivery of these carriers and their anti-pollen barrier responses, they have many disadvantages. it is known, for example, that the ozone antigen-speelSkus tmmune response can be expressed (epli-sappress) by the presence of antibodies directed against the carrier, in this case tetasostostin, [Di Jóim et al., Chain 2, 1415-141S (19 & 9}). j) In humans, a high percentage of the population of nuggets has a pre-existing immunity against DT and TT because they are immunized untypically with these antigens.In the United Kingdom, for example, 95% of your child receives DTF octof Other authors have also described the peptide-ohpaayago problem in animal models (Sad et al., pp. 74-225-227 (1997)); Schutze et al., J. Immunol. 2519-2322 (-WS

In addition, in the case of vaccines that require regular confirmation, the use of highly immunogenic carriers such as DT and TT is likely! in a manner that suppresses the polysaccharide's lymphatic antibody response after a few injections. This multiple itnmonisation may also be accompanied by unwanted reactions such as the "eagle & yed type hypeíresponsiveness" (DTH).

ΚΤ, ismert is known to be a potent immunogen and has been used as a carrier in IgE papain-linked clinical trials. However, some adverse reactions (DTB-like reactions, or IgE-sensitive latex), as well as anti-viral antibody reactants have also been observed.

Thus, the selection of carrier proteins for polysaccharide-based opioids requires a balance between the carrier function in each patient (need for broad MHC recognition, high level of anti-polysaccharide antibody responses, and low antibody response to carrier,

The polysaccharide-d-based oestrogens have many disadvantages. This. this is particularly the case for combmaha vaccines, where the ephop sapper is particularly problematic when the same carrier is used for different bacillus hybrid antigens. A.W0 ^: 98/51339, published as publication; multiple carriers are used in combination vaccines to attempt this grateful gardening.

The present invention provides a live carrier / polypeptide immunoglobulin immunoglobulin with a carrier that does not suffer from the aforementioned disadvantages of the body.

The subject matter of the wood A. / faswtopMítí protein £> Ocho (European Patent Application No. B 0 594 4W B) or a hog-segment polyseband base thereof is an immanogenic preparation, including ohoThe following applies to the phthalococcal bacillary vaccines.

Taiohimahhi discusses: comprising: an oleaginous composition which comprises at least one (preferably water-soluble) Sb'ppnwsmns pscmmmka; polysacchartd-antigéoh and Sb-gpfemzw pvmmsm? me phyto-weekly antigen, or its Immanologically textual equivalents, with Thi-adjovshs (an adjuvant that has changed Thi tnrntnn's response). Preferably, the vaccine composition comprises both the polyhematic protein and the h1l adjuvant. The compositions of the present invention are particularly suitable for the treatment of senile polio.

Pneumococcal polysmchmid precursors (conjugate cleavers) may not be able to protect the pneumococcal lesion in the elderly peptide, in which the incidence of this disease is very high. caused by the production of antibodies against the polysaccharide polysaccharide, which eventually results in hygromycin iagocytosis), however, the portions of the participating opoalcic meohsoismofc do not function either. in the elderly, i.e., the production of polymorphic nucleus cells ("polymotphtmnelear celT", PMN), the production of sapapoxide, the production of other reactive omega species, the mobilization of FMN, BM19 spopiosis, PMN defibrillation, Asa antibody responses are also defective.

Normal! Contrary to accepted dogma, the normal sslpt of anti-envelope-poltssachand antibodies is somebody trusts me to be effective in clearing my bacterium completely, and my pnepmokokkusz ^ tzdaseoktskr can be attacked, avoiding? it feels like a twig.

Surprisingly, it has been found that the simultaneous stimulation of the iris-mediated cell-mediated yoke (e.g., T-cell-mediated petunia) along the hypermalid branch of the pathway (8-celled SM-mediated) results in a slagergic effect, which is able to they This is a recognition that would assist in the PNO-mtokokkusz fertbzes prevention (or treatment in general, but is particularly important in preventing pneumonia Lobo (cut · ^- treatment) in the elderly, in whom the pídlszacharíd based oliöányagok not effective.

It has been found that both branches of the ileum system can synergize in this way when (preferably conjugated) with a pneumococcal polysaccharide pneumococcal protein (preferably expressed on the surface of the pneumococcal, or secreted or released into the ectoplasm) and processed. MHC contingent). Although & pneumococcal protein may suppress cellular immunity by itself, it has been found that the presence of a Th1-inducing adjuvant in the olefinic formulation aids this branch of the immune system, and surprisingly causes the two branches of the immune system to synergize.

The following X, selected pnssmocks, two baconfesrtd conjugate residues were primed with dö-MEt.

The present invention also relates to an antigenic composition comprising one or more pimnechemical polysabaride co-gametes. Packaged with 3D-MPL adjuvant, it is basically an aluminum-based dispenser. wherein at least one pneumatic agent is significantly more immunogenic in 3D-MPL-containing formulations than shotguns, which also contain an adjuvant based on sD-MPL and aiondni.

In preferred embodiments, the present invention relates to antigen-ready forms comprising one or more of the following pseamococcal beta-troch polysaccharides: 4B, 18B, 19F ss 22F smtpspps. In such formulations, each of the polysaccharides is surprisingly more mature in formulations containing AfAMPL alone, compared to 3D-MFM and adjuvant based on ahrrninirnn.

Thus, in one embodiment, the invention relates to an antigen composition comprising serum spp.,? 8C, 19E, or 23E conjugated to a tmrmmogenic protein, a bsm-bsahearm, and a 3DMPL adjuvant, wherein the composition is vm -based adjuvas.

In a second embodiment, the present invention provides a combined antigen composition which is substantially free of alntnin-nm-based adjuvant and which is 3D-MPL adjuvanted and contains two or more pneumococcal bttrocpolsisehartd conjugates comprising 4, , Any of the serotypes J9F or 23F.

The following relates to an embodiment comprising bacterial pseudotannel pnotoin B &lt; 3 &gt;

The present invention relates to a polysaccharide germline antigen which comprises a pleural non-carbide antigen derived from a pathogenic bacilli, which is conjugated to its factor. The invention of the present invention is polivaleas compositions, wherein a vial is? polysaccharide antigen for protein D is coagulated with iron.

The following relates to Embodiment 1, comprising pseudomococcal polybasebaride resuscitation,

The present invention is an improved vaccine. Advantageous® for the prevention and amelioration of pneumococcal phenosis in the elderly (and / or in the pediatric population).

For the purposes of the present invention, a patient is considered to be elderly if he is 35 years of age or older, typically he is 1 year or older, and more generally 65 years or older,

Thus, a WgysMsöássi method of the invention is provided by a vaccine composition suitable for use in the elderly (and / or "semen and infants"), which is at least one 3rrepmeoec. a polysebbarid acyl gene, and at least one 'poemnömue' protein gene.

A second preferred method of the present invention is a ssny brain composition (suitable for preventing pneumonia in the elderly) comprising at least four germ-free / polysaccharide-containing, at least one anti-inflammatory, and at least one anti-inflammatory agent.

It is foreseeable that the vaccine may: be suitable for the treatment of pneumococcal syndromes (such as stone-laceration) in other high coagulation groups in the population such as infants and young children,

In a third embodiment of the invention, the present invention provides an oleaginous composition comprising a pneumococcal polysaccharide antigen and a Thi adjuvant.

/ 1 Zg / d / wnfrn 'seeAa.'j Őfreumc.vcoio pfnejmoume jSjoffeassfegriflf- ^ Hr / aié ^ á

Typically, the Strcococeas vaccine (ciburosen conjugates) of the invention may contain palsahydride antigens. where a, bosaccharides are counted from at least 4 paewekokkasx love samples. Preferably, the four serotypes are his. 4, 19F and 23F. More preferably, at least 7 strobes w in the composition, for example, 4,6B, 9V. 14. derived from serotypes tdc, 19F and 23F. More preferably, at least one serotype is present. the. In one embodiment, for example, the composition comprises: (preferably conjugated) htuofisilic anhydrides derived from serotypes 1, 3, 4, 5, 6B, 7F, 14, 13C, 19F, a preferred embodiment of the invention. There are at least 13 (preferably conjugated) gollsaehydride tigagens in the ready-to-use, additional solisephephenic antigens, such as 23-vulva (e.g. 1,2,3,4,6,6,7F, 8,9N, OV). Compositions 10A, 1IA, 12F, 14, 158, 17F, 18C, 19A, 19F, 20.22F, 23F and 33) are also within the scope of the invention.

For immunization of the elderly (e.g., prevention of pneumonia), it is preferable to include serotypes 8 and I2F (and most preferably serotypes 15 and 22) in the 11-valent vaccine disclosed above, while in infants or toddlers (where ), preferably serotypes bA and 19A are used to form a 13-valent vaccine.

For the prevention and amelioration of pneumonia in the elderly (aged 55 years and older) popliteal infants (IS to one month) and infants (typically IS months to 5 years), a preferred embodiment of the present invention is the disclosed multidrug-infested polysaccharide / wxw.

Sfeeptoeomri overhead or arrnak. it is feminine with the hmccronical equivalence of humidity.

In the apartment report, a "peptide of transgenic equine stunt" is a peptide of a protein that infects at least one epitype of the supernatant. llyea epiphytes are characterized by high purity, high potency and ability to replace bacterial killer antibody response or to prevent toxic effects in the host. Functional exogenous protein is more than 15, preferably 31 or more consistent. Most preferably, the protein is a feagmenser, its deletions. as an example! its Irish zmemhran-delenious variants (i.e. & protein extracellularized derivatives), staples, splicing, or genetically deletion derivatives, and the like, may be used with the proviso that the protein is essentially the same Inbnv.

Preferred proteins are those psenmeknkknsz proteins that bind to the outer surface of the pnetmtokefex (the host immune system is able to recognize them for at least part of their life cycle), proteins which; secretions of pttenmokoccus or release, most preferably, protein

SSr ^ ococoas jMeu / wniae toxin. adhesin, a two-component signal transducer, or a propellant, or a functional equivalent thereof to their hymnologiques.

Particularly preferred examples of non-limiting proteins include pneumolysin (preferably chemically or by detoxification by rotation) in such a combination vaccine [Mitehell et al., Nucleic Aeids Knife. 18 ,. 4019, p. (1991)}; Mitekéi! and so on. Einehem. Biophys. Isaiah MA7, 67-72. live. (1989) (WO 96/1) 5859, WO9W8O9M. and International Publication Publication No. WO 99/83884; Variants of PspA and tmssamesnhthan defetah (U.S. Patent No. 5,804,193); Variants of EsgC and Mahsmesnhrass-defetah (WO 97-9994); Variants of PsA and transmembrane delta jerry and Pair®, inf. 64, 5255-5262. old, (I996) j; variants of pneumococci, c-binding proteins, and membrane-t-deletah; Variants of ChpA thymus membrane defects (WO 9741151. WO 99/51266; painters); glyceride b.id-3-fesfate dibenzogenase [infect. immune. 64.3544, p. {1996}]; 1-ISP7Ö (WO 95/49029, International Publication): PcpA (Sanchez-Beato et al., FEMS Mierofeal Lett 164,207-214, vol. 1998) 1; M-like protein, SB patent application, EE δ 837 110; and Adhesive 18627, SB Patent Application, EE 0 834 56.

However, the proteins used are preferably any one of pneamohm, PssA, EspA, PspG, CbpA, or combinations of two or more. The description discloses: immunologically feakslonsife: equivalents of such proteins are defined above in Serbian.

In the composition, the protein may assist in inducing an immune response mediated by T cells against feneutnococcal disease, which is particularly necessary for the protection of pneumonia, which interacts with the Immoral branch of the azin mammalian system to inhibit the pneumococcal inverter and stimulates the opioid.

Further advantages of using a female antigen include the presentation of additional antigens in the form of the phosphatase adpses gr (i. E., A method of treating the amine; if toxin is used).

Brasvk srisgiátakissri brain rssagvakiskáo! ssstsd szsssásss is the subject of assáirsssiasy -Si-iy: ^ · í ::? <. ^ <?? í <yaasvaoMx? ssisóassyaa. orz;> taller bed, sMsryóáür; Ag & ibb lime apsprishosssa formerly fat or sarcasm; ás sa sa M M f M ps ps ol ol ol,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, ag ag ag ag ag ag agasi, ag ag ag ag ag ag ag ag asi asi asi ag ag ag <; · - vioavóssos kotsb Xovyas.zzzv-s y ^! vz.z / v; v, v vásásásás and sartaisssaz. SAsssriok ogytkv vibssyesvss oavassiohzsss or TsasA or PspA or ChpA gkgviooyosabboa dotoysisk grsssissdsSzissy EkvgM; kstrsshksaaeS Agasabs prssssssS'Oitzstsf or Ísksk derivative and PsoA Sssr-ahssaz.

Moss ahvgysa dye aoshiva in the bassoon of ssssasossizas; kaystsoAios are you thinking about brain? that sledge is a ssohshawarAk össssagvkbaa harshness ^ aisroaooaars. Azat. that sbsPAvsvkvshíksk;: - zoss _; for the MAwkioádöksss winemaking hafa kossjagábsApsk. awfyek osotiákM á'Azssvs saggy nybbaaaC Τνζ: dósvök that s stdátsssássy hair; the tdks-Asazoss pohszbakarsáttkA hyoss hordozóíáhásáához kspcsotgrk, Press partUzstohaPd ^ itrsstssssögáiSsk sdösPkssára; doses per ákasássosass askakrisízors: carriers páAós Koza ósMszik the dsrkárA- · digs Tosa sarzáossoA (St. _AT CP.MM7 digs Ti '), körsosásiga (, Tvyh «The kssspsA ) hossaöesassAáa. (K; LH) _S: M í?; V «XyM <k? ÖMPCAíx and ís sahsrkisiiys s; $ zi iroi; AbásjasziSrsssazáka. Sppf)).

Azosrhao ssiesses prodama kotsasobáos azoss slosso used sorss; s-sksdi? Gyrkvve (1See: "Using the ASAsSssssss; S;

Is the iaAfsssássy object and taste? ace; how to skbrybss tssogyssAssessing saltSpreading acid from hssrdsword prsSssohzrkisb & ssss isrisrsrsssssgassing casserole vassal alsace, asrasy ssatss sassvod oozs hsks. Pstastasokokka is a viooyous carrier of oaPsia transfer pídiipk sísshsassrsgáss kassaasaásyvyv (or school supplies) tbazvíyróaar ás / A water? pro-episode D sequences: · (EP 0 Sodium 6; 0 B, ordasa vadasi szishadakssí Irat). or catch me hagravsi !. The aiksbstaAisss _: tssvgAAA isagstsarsook khzá íastazstsk sog stó? -Ság öpsdpvkas: carrier Iroysssoosak EkMyósv ;; a psotoM D skstorasss; s irshas iáAvrsrdrsátss iE clock clock saxhafis.

The písvsssssokokkszsz pabstabasád sovsthbs «Mssyós carrying sssaga the pssassrssoksskksss Ahágz tss oas as & Jhsov atokokkas Abásá á k 'usual dr).

The Says; Say Common Sages Sasa §Assytksazs zstAvta · aisSisso. Magivorous adMvdssss include AssshssMrS-'Sikk, rye yasdasl akssraoisass · Sodrorid gri (atasss) orAstthshísssAeszPsh dv ksst, iron or vs salt also alkarssazhoMos or oak; ssssszssssszsóia.

The sdbsvásss ssssósóososs Ths tlpssss response is blind to the Asstsrsssvkhss siiíEntermediate backs.

áAkórsaA YYR <váALZt "?. yrfetásvyá

TEl aspis sshóksssak srsagao ssssazrarsvivia hsystrssoa kadvozstf throught the soh-public itssssstasvásas asasas assssgár, ro, salt tho-slassassas vkokiossk ssragaS: kisvesstsa

It is important that osssAkazzdssk assta is the abscess of the Thk and Tss-type ssosss-ssss of the statsr. The v & hssógbao brain is bed-like srsssysssísvístasz topos. asvXyvs vfebsozbass. Thá -rssk or ThA srak sshatassk; v. Azrsssbssi: sgrssks'ass tsáisysbsto ;; the obxskihok rsstiárgah sssy sssódoa BgyáAsrshá vorsssk asísss bioasssakss and CotTsssass (Astststai Rsvsosv of fiSriSrssrsoiogy 7, MA p73. ThiOtpsssá vdkssas P · Sssrs Vossi: fNP-y and Η .. · Τ v AskirnvsrssviasvvA kassvo; Sarosat: Other, talk to ThTAiisrsaá irssrsssasiasaavivastava; kozvvSsvssa; Box-type eitokines, η1η, for example, ΙΕ-12 are produced by non-T cells, in contrast to Tb2. Responses (relating to the secretion of 1-4, 1L-5, 11-6 and 11-Jö. Suitable adjuvant systems for eliciting a? Thl-tspase response include monophosphophilyl judipd A and its derivatives, particularly 3-de-Oaetl). -monophospholyl IIpsd A, and moso-bisphenyl lipid A, preferably 3-de-O-αEmnt5 phosphoryl lipid A: cinnamic acid with aluminum salt.

The improved system comprises a combination of monophthalophenylated A and sapoa derivatives, preferably OS2; and 3D-MPL. as disclosed in WO 94/1) 0153, or as a less responsive ready-to-use, wherein QS2i is attenuated by cholesterol according to WO 96/33739. would publish international disclosure, according to document discovery,

Knlöpöscs is an effective adjuvant; WO 95/17218, an arable international publication containing QS2lA, 30O-MFkk and pumpkinfolol, which is a preferred formulation,

Preferably, the vaccine further comprises a saponin, more preferably QS21-. The formulation may advantageously be formulated with an oil-in-water emulsion and a formulation according to WO 95/17218.

The present invention also relates to a process for the preparation of a formulation of ohoic substances. according to which the protein of the saccharide is mixed with an excipient binder such as a 3D «MPL« signal,

Oligonucleotides containing methyl Cp <3 ~ (WO 96/02555) are preferred as a supernatant for Th1-type immune response, and are suitable for the calirification of the invention.

Particularly preferred nests of the present invention include one or more of the costumers, one or more pneumococci. Protein and W adjuvant The induction of intercellular WtWd response by pneumococcal protein (as described above) and coexistence of the two branches of the snmrn system are facilitated by the use of such Thl against.

According to a further embodiment of the invention, the present invention relates to an Immunogenic Desire Vaccine for use as a medicament.

Yet another aspect of the present invention is to provide a composition comprising a polynuclear polysteroid co-formate and a Thl adjuvant (preferably 3 © -MP: t) capable of inducing a drug conversion or bwörab antibody response. asa in the pope of the unresponsive,

8-38W of the population do not react in a known manner; pollsadharyl (not reacting to more than 5% of the serotypes in the vaccine) ÍKonradsett et al., Scaná. J. immunized. 48: 251 (1994); Rodriguez et al. (1996) JIO 173, 1347, p. It can also be dehydrated by conjugated vaccines [Moshez et al., Clin, Int, Dis. 27, p. <1998) first This can be especially serious in the high-risk areas of the popcorn (infants, toddlers, and the elderly).

It: the combination of fecal, predominantly conjugated pneaokoxocollistoride (which is able to respond to abesosy in a particular population) and a Tbl adjuvant (see section "Tb I adjuvants of the invention") may surprisingly overcome this aasmic reaction. Preferably, 3D, MPI .. should be used, most preferably 3D-MPi .. without an amylamine-based adjuvant (which gives an even better answer). The present invention thus relates to such kits, and further to the Thi adjuvant. is the use of the pof ^ charid adgom® not responsive to treating or protecting against psoriatic mucosa disease, as a suitable cure for cosmic gheefeakkm polysaccharide amulogeumecol? manufacture.

In a Bgy embodiment, a method of preventing pneumonia or treating pneumonia is a method of administering a safe and effective amount of a vaccine. as described in δ to an elderly patient comprising a vaccine comprising Ssrajo / oeoceasr pHeififiaiae polysaccharide and containing either I'hl adjuvant or pennenum stone protein (preferably both).

A further embodiment of the invention provides a method of preventing or preventing inflammation in infants or toddlers, comprising administering to the infant or toddler a safe and effective amount of the vaccine, which vaccine is 5 &gt; epiloc <? E <w jwwm póh. . and either Wi-adjsVan or ptwococcal protein (preferably both).

In the above inventories, the polysaccharide androgen is preferably present in the form of pseudobaric-whey-bean anthers: ksni.

More particularly, the invention relates to a method for protecting or treating a mammal susceptible to infection by administering the brown substance: srisristna or yattearyl. These are among the modes of administration. intramuscular, glial peritoneal, istradermab or subcutaneous injections; or mucosal administration to the oral, intestinal, respiratory, or coals. Intra-axial administration is advantageous for the treatment of pneumonia or otitis media (since the nasopharyngeal phthalococcal delivery is more effectively prevented, so that infection can be attenuated at the earliest stage).

The conjugate antigen; The amount of each dose should be selected for each vaccine, so as to select a protective immune response in typical vaccines, significant adverse effects may occur, and the amount may vary depending on the specific hmnatogen used and the presentation. In general, it is expected that each dose will contain AU-W ng of the polysaccharide, preferably 0.1 to 50 µg, more preferably ≥ 1.0 to 10 ngmt, most preferably 1-5 ng

Áz- fehérjetsrtalnn ^The vaccine of the & PKNS: IO8 be Í ~ ng, preferably S-SO gg> legíipikossbhan $ 5-2 pg.

The optimal amount of the components of a given vaccine is routinely evaluated by routine assays involving the subject's rabbit immunization. Following Primary Immunization: subjects may receive one or more booster immunizations as an adjunct.

Generally, vaccine production is described in "Yaccme Design the Subunit and Admantant Approach" by Power & U and Newman, eds .; Revealed by Pfenura Press NY (199S). Reveals Fallerton Closure to Ixposomes (§ 4,235, 27, U.S. Pat. No. 2).

The following are the 2 'selected pnococcal polysaccharide conjugates and 2D-MPL. compositions comprising compositions.

In the context of the present invention, the term "pneumococcal polysaccharide conjugates of the invention" is intended to mean a sheath of Srrep / ococcus pwomowoe envelope-polis2ae-sdok, which is more immoral in kits containing JD-MPh. like 3fe-lriPh in aluminumϊ · *>

2.Ζ add the adexeants to one of the finished dishes (example 4, BB> 18C, 1EF, or 23E sserodpas).

In the context of the present invention, the term "essentially," under the term "dwsmnn-i-afepa: adjuvant now" is a composition! which does not contain sufficient auxiliary-based adjuvant (e.g., amino acid hydroxide), and preferably sphymoma, to produce any reduction in the polynuclear polysaccharides of the present invention by any amount equivalent to the composition. , which is a 3D ~ MPL-iartslin without added aluminum-based ttdjuvant. Preferably, the Igstigen-cooled kit should contain an adjuvant substantially containing 315-MPE. A. The amount of adjuvant added per dose is preferably less than SG pg, more preferably less than 3µg, more preferably less than 10µg, and most preferably no adjuvant is added to the antigenic composition of the present invention.

For the purposes of the present invention, the determination of whether a polysubborn conjugate of a primer cocktail is significantly increased by 3D-MPl. preparation slmgmfekátw! immunogenehb, when containing only 30 µM-MPE alone, the ratio of GMC µCi to keno-center containing the unique Gfe-MPk and containing 3D-MPk in aluminum-adjuvant adjuvant} must be greater than: 2 » _; preferably mini 5, more preferably ntbA 7, more preferably reed 9, and most preferably miter 14,

Problems with the polysaccharide approach to immunization include the fact that the phobebaebides themselves are weak nnnmnogens. Strategies designed to overcome this deficiency in tmmunogenicity include: b &apos; adjuncts to large unraunogenic carrier proteins, which nJ.eke «T-scuex help nvjtarx \ F'tmyós. that. » The pseudo-carbides used according to Thulman are coupled to a carrier protein and provide an ancillary T-cell help. examples of such carriers which may be used include dorsal, mutant diphtheria and tetanus tubboids (DT, DT CRbiiP? and TT), hornbeam pigs (, Aeyhole lymphatic) hemcctaniosis (knife), tuberculin purified protein derivative (PPD), and, Vg «-Comb« OMPC,

Most preferably, the protein D protein of (y / ueHzaa) European Patent Application No. PPG 594,610 No. 8 or its self-segmented & aim & zzuhfe (see the following section for a further embodiment of the invention containing bacferal polyssehadd-profeb D). The dressing gowns of the present invention are refined.

In one embodiment, the anti-angiogenic composition of the invention is formulated in a polynucleotide polysaccharide serosipnsf (PS1) conjugated to a femnogeneic protein and 3D-MPL adjuvant, whereby the preparation is substantially free of any other form of adjuvant. , PS ISC, PS IPE: or Pb 23F, conjugated to an immunogen protein and formulated in 3D-MPL adjuvant, wherein the preparation is substantially free of the parent compound.

In a further embodiment, the present invention relates to a combination antigenic composition comprising two or more pneumococcal polysat & arid water jugules of ES 4, PS 68, PS 1SC, PS 19P and PS 23F formulated in 3D-MPL adjuvant, wherein the composition is essentially alam in intn-based adjnvv evening 1 free.

The pneumococcal polysaccharide conjugate of the present invention has been significantly influenced by its combination with other pneumococcal polysaccharide stone jugates since immunization (see Example 3). Accordingly, in a preferred embodiment, the present invention relates to a combination allogeneic composition comprising one or more polysaccharide co-polysehydes of the invention, further invented by means of a pneumococcal polysaccharide-containing drug, wherein in general, it is free of adjuvant.

In yet other preferred embodiments, the present invention relates to a combination antigenic composition comprising at least one, but preferably 2, 3, 4, Desire all PS 5, PS 6B, PS 1 SC, PS 19P, and FS polysaccharide conjugates. 23F Circillium and, in addition, any combination of other pneumococcal polysaccharideAonjugan, wherein the composition is formulated with a 3D-MPE adjuvant but substantially free of aluminum-based adjuvant,

Typical conjugated polysaccharide antigens of the present invention are typically conjugated to polysaccharide antigens of at least four beta, eleven, bzenephem, fifteen, or twenty three serotypes (see Structure A). from a disease to be treated

The sariginous kdsritaen of the present invention is preferably used in vaccine formulations for the prevention (or treatment) of pseudococci infections, especially in the elderly, infants and young children.

Other embodiments of the invention are common; providing paint asphaltic preparations for medical use; and the use of one of the above antigenic formulations in the manufacture of a medicament for the prophylaxis (or treatment) of pennenococcal disease.

For the treatment of infertility in the elderly population (<55 years) and in middle infections (infants up to IS month) and infants (typically from 5 to 5 years old), a further preferred embodiment is the discovery of a mammalian polysaccharide or a combination thereof with an inorganic black &lt; RTI ID = 0.0 &gt; &lt; / RTI &gt; equivalent &lt; / RTI &gt;

The additional cost to date is that kits (and vaccines) are preferably lyophilized up to use, when they are reconstituted with solvent, most preferably, they are bolylated in the presence of HRD-MPL and re-substituted with salt.

The lyophilization of the formulations results in a more stable formulation (e.g., it prevents the degradation of the polysaccharide amigenes). Surprisingly, the process produces an even higher level of antibody against pneumococcal polysaccharides. Particularly insignificantly, they hizoayuh the PSBB conjugates. Thus, another embodiment of the present invention is a lyophilizate containing PS SS kinase _{: an} overgene formulation without 3D-MPL adjuvant and essentially an aluminum-based adjuvant salt.

For more details on the inoculation of vaccines, see the section on "Invention vaccine preparation according to the present invention.

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. ·· '.?: L-Aassöeisaseilla-sda-spadhakse-setsasdiaraberA (GDAd) ess ·; bálé ceagcsssi esössydsez: alkah · madzssk pss? IsksAsAiÖ-whiteness) »AeppsváWaök satdiséee; The siandAo Reaction page is acidic among acid sponges Happy people have succeeded with the alaskan ρii ··· javékeay peaseaehassdok Ase Esess seseezes lePdevé; ss hahezélkeéé ss-sés köayedaa ka |> öi> dlat,

Piddepinshid'idys is a fluid; xpgy ssiekkildssp precursors GDAP-b is synthonithiolxsp elppA lel, and SkOAsal in pelssexa-kl-alkaliide A) -A. CDaP's Bubble Shape Reactor Balvsd Responds to Starting and Stepping Out Asa ess ars; add the horiieadrshés to the seak Uéeisgy .. liaasek eeesse · esssspöi'isas seaaélp-sk is the active blind pislssaseisandésla fossil sa;? araa-kikésr ajksdva,

Following coupling reaction A, a large excess of glycine is added to bind the residual activated groups. The product is then gelatinized to remove unreacted carrier weights and residual reagents. Accordingly, the present invention relates to a process for the cleavage of a polysaccharide D conjugate, wherein the polysaccharide is activated and coupled to protein D.

In a preferred embodiment of the invention, the present invention relates to a lozenge preparation (or vaccine) formulation comprising Sn-ewireca®.jmemomea? infections.

The germs by which gneisses reach the lungs, spinal fluid and blood are barely perceptible. The growth of bacteria that reaches normal lung bladders is inhibited by their relative dryness and the macular activity of the pleura. Any anatomical or physiological change to alter these coordinated protective mechanism mechanisms & susceptibility to pulmonary infection. (1997) 1,

Typically, the Streptomycetes counterpart of the present invention contains a proprietary D-ring helix that is derived from at least four, seven, eleven, thirteen, fifteen, or thesaurus species. See the section on polysaccharide genes of Streptococcus pneumoniae according to the present invention, which is a preferred combative of feuta serotypes depending on the disease being treated.

In another embodiment of the invention, the present invention relates to a hepatitis vaccine; Preferably the A, C, WI35 and ^Y szsrodpusökbői, one reason feglönídsabb Afebsmoa bacterial agyháriyágyuiladás. Carbohydrate harris in fall organisms can act as determinants of the carbohydrate and is the target of protective sweets. Carbohydrates are over! well-known heptal hogen halogens in fttsd fetuses, A. is a suitable spermatozoa for these polysacfearides, protein D, which provides T-site epitopes that can activate: and .mduking the immuneldgtai .memory,

According to another embodiment of the invention, the present invention relates to a Bemflox mfe fee8smé b burek pöltszaolwid (PtePj-profein 13 sputum),

The tsíálmáay infect contemplates combination vaccines _ls>: range of different pathogens for which immunity Elten oyüitansk the protein D carrier is surprisingly suitability elíéattyagökhan combination as a carrier, wherein more pohszueharíd konjagábnk antigen. As mentioned above, the occurrence of superstructure suppository is likely to be the same when used for the same type of substrate erythematosus, WGW51.339. would publish internationally; discloses formulations which attempt to minimize this interfering cooling by conjugating a portion of the prepared pellet SdvA P &&quot; to DT, the remainder to TT.

Surprisingly, it has been found that protein D is particularly suitable for minimizing such epilepsy suppressive effects in combination vaccines. In one combination, one or more polysaccharides can be advantageously conjugated to protein FJ, and preferably all antigens are conjugated to protein D hs. Such a combination sbówag ^ bas A preferred combination comprises a vaccine. which protects against infection by the C and Y (and preferably A) serpentine of Yebserfe mmfepgoles, wherein four or more polysaccharide antigens of serotype Y and C (and most preferably .A) are in protein D. linked.

// <zamopathic pollsaharide-based vaccine (PRP is preferentially conjugated to d'T,

DT or CRM197, or most preferably, protein D) can also be formulated with the above combined ingredients.

Many pediatric vaccines are combined; are now being given as vaccines. to reduce the number of vaccines to be given to the child. Therefore, other pediatric excipients may be formulated with the vaccines of the present invention. For example, the vaccines of the invention may be formulated or administered separately but simultaneously with well-known trivalent, inoculated vaccines that contain dermal toxin (DT). TETA "to" 4oxoidbt (TT) and penusssász kötuponenst [typically detoxíükáít psrtPSsztsz toxoid (SD)], and filamentous hsnsagglntlniní (FHA) comprise optional psrtacsm & i (PRN) and / or aggluüninnel, e.g., the commercially available (NFANTRlX 'DTPa ^rM (SmithKbneSeechasn Dfelögieais), which contains the DT, TT, PT, FHA and PRN antigens in a phage or whole cell pertussis component, such as TritAnrix ™, a commercially available vaccine from SnYtltKlineBeccham Bsologscals, may also contain an antigen other than exemplary hepatitis B antigen. , polysviral aniline gene genes (exemplary inactivated Trismlene puli © vtres ~ IFV), olfermerre / transfeuerial membrane proteins, non-typable tigmnpbóW fepsrerew febenes, M white blood cells R.

Examples of preferred calendula whitegerms which combinase. in vaccine lobes: (especially for prevention of midbrain): OMP106 [WO 97/4173 L (Aníex) and WO 96/34960. Arable International Publication Publication (PMC) I; LbpA and LbpS [WO 98/55606. International Publication Publication (PMC) 1; TpbA and TpbB [WO 97 '] 785. es WO 97/32989. pledge International Publication Letter (PMC) j; Copö (Heimaten et al., Intect. Homem. Five, 1993, pp. 29-2000, 1993); UApAl / 2 (WO93 / 03701, International Field Publication (Xlnfeersky of Texas® and ÖrnpCO, Non-typable tfamopAdvs? * $% &&&& antigens that may be in a combined vaccine (especially to prevent otitis media): fhrhrfe protein [5,766 bulls, US patent (Öhio Slafe Researe.h Foundation)] and fusions of our pepitase (e.g. Peptide Tufts, U.S. Pat. No. 5,843,464, U.S. Pat. No. 5,843,464 (International Publication No. ÖSUj or WO 9W64067); OMP26 (WO 97/01638, Dehydrating International Publication (Cortecs)); Po [European Patent Publication No. EF281,673]. State Umversitv of New York}; TbpA and TbpB; Hsa; Mmwl, 2; Hap; and Walnut,

Preferred pediatric vaccines within the scope of the invention are:

a) M C polysaccharide cortlogate: and / Msmpp & lpicAlcic polysaccharide conjugate, optionally, Y. With a polysaccharide conjugate of Es or Y provided that at least one polysaccharide atigen is present, and preferably all protein O is lubricated with a.

b) / fe vaccine according to a), plus DT, TT, the pertussis component (preferably PT, FIM and PRN), bepat His B stimulator and IPY (inactivated trivalent polyvinyl vaccine).

s). pseudomorphism; polysaccharide-ziggenic protein to D for yoga,

d) The vaccine of (e) plus one or more Brarlb mimic-Malss and / or non-typeable J / l / l6 / l lubricant antigen.

All of the above combination vaccines may benefit from the use of a proton D carrier. Obvious! the more media we use in a combo? oiroaxtyagbar, to avoid eoep-op suppression), aona is more expensive and-ohyoooit3.hh is the final © Crab. Conjugation of all or all of the polysaccharide antigens of the cobin vaccine to protein D thus has a significant advantage in preventing pneumonia <55 belts in the popliteal cavity: prevention of infertility in infants, or in M young children. a method of combining the conjugates of the excised mucinous titer cocci with the stear Dokassas-Dok-Proem D protein with StM / xmeoccHs iwtiw protein, or an immunolegially tissue-like equivalent thereof. See "The Invention Safety Pneumococcal Proteins * section above for the preferred proteins and protein combinations that are used in such combinations,

Accordingly, the present invention relates to an immunogenic composition. which J ^ öcombywamös & e. polysephearide protein D conjugate and hemp / oohee », '? Wiostöe contains white antigen.

Preferably, the polysaccharide-profeb D coactivator D according to the invention is adjuvanted together with the vaccine kits of the present invention. Suitable adjuvants include aluminum salts such as scintillation gel (altira) or ·· ainminuts phosphate, but salts of calcium, iron or zinc, or acetylated lirosine or acetylated polysaccharide or abionose may be used.

For aged © materials, it is preferred that the adjuvanted Thl-type response is the preferred inducer.

For more details on Thl adjuvant salts, see the "Thl adjuvant salts of this invention."

According to another embodiment of the present invention, there is provided an imhthmogenic or vaccine disclosed for use in medicine,

For the preparation and synthesis of conjugate tunate A, see the above section "Invention of the invention from a synthetic material prepm".

Protein EM is also advantageously used in the midgut vaccine because ppm is a fanmunogen capable of inducing B cell-mediated protection on the non-typeable Bhmw / glial gland. Ntfil can flood you. host cells, and the effects of B-cells mediated by the breeder. Surprisingly, they display sg) atal for increasing the potency of protein D: (either alone or as a carrier for polysulfearla) as an antigenic agent for otitis media. It was meant that co-administered with the LM adjuvants of the protein by inducing a strong Th1 response in the subject by opnmalizing the cell-mediated branch of the immune system against protein. Surprisingly, this was achieved using a lyophilized formulation containing protein 5 and Th 1 adjuvant (preferably 3D-MPL), which was reconstituted shortly before administration. e, ams kw <jmtvk produced it? lyophilisation of tp.times.times.1 and TbI-adjuvantstillaryl and the preparation of such preparations. Intermediate & managing: to handle.

In a broader sense, it is possible that hothylation of an immortogen in the presence of Th1 adjuvant (see section "Thl adinvants according to the Satama"), preferably in the presence of 313-MBL, generally enhances the immune response to the immunogen. The invention is applicable to any immune gene that requires a stronger Th1 immune response. Such anmunogens include bacterial, viral and tumorigenic protein genes, as well as salivary genes; proteins and peptides taralalmsKbak,

Patent Examples

The following examples illustrate the invention in more detail, but are not to be construed as limiting the scope thereof.

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The U-v & lens vaccine candidate includes ISCs I, 3, 4, 5, 68, 7F, 9V, 14. Seroprotein bprok polysaccharides 19F and 23F, which have been prepared essentially as disclosed in European Patent Application EP 72S {3. All polish bridges were activated and modified using CDAP chemistry (International Publication No. WO 95208348) and incorporated into its host bar. All polysaccharides are conjugated in their native farms, except for the 3h serotype (which has a size of 77 / ozA-í / 444 / * *, ·

The selected box stomach mite protein D m can be typed from tfaeampOw hgTaers »expressed in eoA.

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Protein D is conserved in all vascular typhoid and non-typeable strains of tf influenza A, a DNA vector sequencing pNIC348 containing the complete protein O gene from Dr A, Forsgreen (Department of Medical Microbiology, University of Malmö, General Ftosp, Malmö, Sweden). See the DNA sequence of protein D and see. [Wall. Tapas. 59, 119-125. p. (1991)].

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A. pMull extracellular capsule is a sledge pSR322 (Cröss et al., 1985), in which transcriptional and translational regulatory elements derived from bacteriophage D have been incorporated (Schatztsan et al., 1983).

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The AR38 A eoA strain was produced by transduction with N99 PlAag, and the strain was previously cultivated on SASÖÖ (galE; TN10, lambdaKiF C857 Alii). Old eoZ from N99 and SA50Ö from Dr. Martin Rosenberg's National Insdtute of Health Laboratory? strains.

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To produce protein D, the DNA encoding the protein is cloned into a pMG1 expression vector. It uses signals from this plasmid DNA to direct transcription and translation of inserted nerve genes. The vector PL promoter is a kill operator and two uses; (Null. and NuR) contain the transcriptional polarity to mitigate effects when using the N-protein (Gross et al., 19S5). Vectors containing the FL pronoun are introduced into a lysogen cob host to stabilize the plasmid DNA. Lysogenic host strains contain replication: Io-deficient lambda phage DNA integrated into the genome (Schatzman et al., 1982). The chromosomal DNA directs the synthesis of the α1 repressor site, which binds to the ÖE repressor vector of the vector and prevents the binding of RNA polymerase to the PL primer, and contains the expression gene for the gene for ^: feeülmt? Thus, PL-opened transcription can be regulated by temperature switching, i.e., raising the temperature of the culture, inactivating the repressor and inhibiting foreign protein synthesis. This expression system allows for the controlled )

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The users of the protein D carrier tenne.exers at A &58; The cpA strain is a derivative of the standard NIH £ 100 N99 KI2 strain (T \ u galK.2 iacZ thr). Incorrect lysogen in lamb contains phage (galE :: TNIÖ, lambd & Kír CÍ857 AH 1) Λ KT 'phenor.pu.- prevents. disabling the host macromolecular simulation. The e857 mutation causes ci-represscr heat-sensitive susceptibility disruption, the ΔΗ1 deletion edge misfold of the right operon of the lambda phage and the host bio, uvr3 and ehi.A locus arm, A: AR58 £, eoö strain with N99 ΡΡdj, previously from SASOÖ? '. cultured (galE :: TN10, lambda & c! 857 ΔΗ1). The insertion of the defective lysogen N99-bc is selected by tetramkhoen via the TNIE-raspposon signaling gene encoding terracycline-resistance in the adjacent galE gene.

The pMG1 vector containing the gene encoding the non-structural Si protein of inilaenzavtrus fpMGNS1) was used to construct pMGMPPPrD. The protein D gene was amplified by PCR with PCR primers containing pHC348vsktorho (Janson et al., 1991) at the 5 'and 3' ends, respectively, of Neo 1 and Xba 1 restriction sites. The Nco I / Xba I fragment is then inserted into pMGNA 1 up to the Nco 1 and Xba 1 sites, thereby generating a fusion protein containing 81 amino acids of the S1 protein, protein D. I labeled this vector as pMGNS1 PrD.

Based on the construct disclosed above, the final construct for protein D expression was prepared. An 8; so-called Bi / Sam Hl iris master was removed from pMGNS 1 Fro. This removes its MSI coding region, except for the first three amino acids of the E1-terminal. Following the vector ligation, the following gene encoding the N-terminal amino acid amino acid fusion protein is introduced:

—MDF SSHSSNMANT ------- NS5 Protein D

The protein does not contain a leader peptide or H-tertiary protein to which lipids are attached aortically. Thus, the protein is not expressed at periplasmic expression, nor is it converted to ilpidem, but remains in soluble form.

Constructs of the final mPG-MDFPrD were introduced into the host strain AR58 by heat shock at 37'C. Bacteria containing the plasmid were selected in the presence of cassamim. The presence of a DNA probe containing protein D was confirmed by digestion of isolated plasmld DNA with selected esdonucleases. The fskombiuAüs £, oo / r strain is called EGD-1,

Protein D expression is a. lomEda PL Promoter Controlled by Ancient ÖE Operator The host strain AR58 contains a temperature-sensitive address, rw.j barrier at kmbda Irt, »oi \ aio, \ pressziol aia, through binding to .giA. Once the temperature is raised, cf is released by Okho and protein D is expressed. At the end of the stain, the cells are concentrated and frozen.

Extraction of harvested cells and protein D flsAylation were performed as follows. The frozen pellet was thawed and re sejttcttyészef fólszttszpesdáituk sejtíeitárő solution (citráípuffer ^pH: - ^'; B, C) to OD "eighth to one tenth« 6o. The slurry was pressed on a homogeneous tatator under pressure at 1000 bar. Homogenization of the cell culture. centrifttgálássa! were clarified and the cerebellum was removed by filtration. In the first purification step, the hair was applied to a non-volatile karyon exchange chromatography column (Sf Sepftörose Fost Fow) by tel. Protein D binds to the column by ionic interaction and elutes by stepwise increasing the ionic strength of the elution buffer.

In a second purification step, the impurities were retained with a nonionic exchange rotogravure (g ^ jferose Fdrr F & nvj charge. Protein D is not bound to the charge, and to the Sroliyah.

In both chromatographic steps the iris salt salinity was monitored by GD. The overlay of the anlon separator column chalk topography, which contains the purified protein D, was refluxed.

The protein D-containing altrafilter filter was finally filtered through a 5 µm membrane.

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The activation and coupling conditions are specific to each polysaccharide. These were challenged with native pelvis hybrids (except PS3} 2M N &lt; RTI ID = 0.0 &gt; Cl &lt; / RTI &gt; bad or dissolved in water for isotecation. Optimal PBS was evaluated for all serotypes.

C? AP (? CDAP / PS ratio? 0.5 µg / mg) of? 0 mg / µl aortic strain intestine was added to the polysaccharide solution. After 105 minutes, a 0.2 M trellial plug was added to achieve splenic activation pH. Polysaccharide activation was performed on this apphine for 2 minutes at 25'C. Protein D (amount from baseline PWD ratio) was added to the activated polysaccharide and the coupling reaction was performed at specific pH for 1 hour. This time, the reaction was stopped with ghsbnet for 30 minutes at 25'C and overnight at dte-O,

The conjugates were purified with gel, equilibrated with 0.2 M NaCl, WO? using a gel filtration column.

The carbohydrate and protein content of the fractions were determined. The fractions containing the conjugate were pooled and sterile-filtered with a sterilization membrane of 0.22 µm. The FS1 / protein ratio of the conjugate prepttrates was determined.

Pure cell culture inhibitors were characterized and met the requirements of Table 2. Polysucbat-id-content in the sky / ml} resorrinol assays were measured for protein content (pg / ml) by the Lowrv assay. The resulting PS / protein ratio (w / w) was determined by the ratio of concentrations.

Activation of polysaccharide by CDAF builds on the polysaccharide and DMA? (4-dimethylammopyridine) is released. The residual DMAP content is specific. SB was determined by an advanced assay.

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Co: sjss.gatts Muscles stored at 4X and held at 3 ° C for 7 days were determined from the supernatant supernatant by supernatant with α-PO antibodies and saturated ansmonone-ssns.

The free polysaccharide: tt-PDAt-PS Enzyme Immunoassay (ELISA) is a sicalotase; Deficiency of the conjugate inhibitor was also measured by n-PDltr-PS ELISA. The reduction of free polysaccharide levels results in an improved boning vaccine.

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Similar assays were analyzed for andgenic whites by the sandwich-EUSA assay, in which capture and detection antibodies alternated between o-P5 and u-FD.

Free Reverse Content (%);

"Free residual protein D" was determined using SDS treatment of the sample and routine care. The conjugate was heated for 10 minutes at 10 ° C in the presence of 0.1% SDS and applied to a SEI-1-PLC gel column (ASTIL 3: 7-Ph <7>). Because of the diameter of protein D, there is a risk of overestimating the level of "free" protein D by dissociation of the structure by SDS.

Sample size determination was performed on a -SEOfIPPLC gel column (21SA overnight-FhWe).

Stability Δ was measured on a SEC-HPLC gel filtration column (ΓΧ &lt; RTI ID = 0.0 &gt; night-Fd'A &lt; / RTI &gt;) using 4'C-conjugated conjugates for 37 days.

Characteristics of the Atl-valsns vaccine.

The protein soluble inhibitors can be absorbed onto the uimsdmtmt membrane and sealed to form the finished vaccine, and, ummmgene conjugates have been prepared that have been found to be the most promising vaccine components. The above improved (optimized) CDAP-coated pneumococcal polysehydride corugates are independent of carrier fibrosis. but preferably with protein D) thus form a further embodiment of the invention.

Example 2 - .feyífotí give me a protest? the effect of snk on the tootslm of IMwlétss psettmokoldens FSFl>

Rat subjects were immunized with Immanizable Shells-Vsfests pnsomokokins PS-PD vaccine at a dose of 0.2 µg of each of the polysaccharides (prepared as described in Example 1) and adjuvanted as follows: None, AlPlX, 3D-MPL. 3D-MFL on AIPCh.

The formulation containing JD-MPL alone was statistically (and bile bilayer) more inmnogenic (highest GMC IgG) than the other formulations out of 11 for 5 antigens; This is true for all high, low-level 3D-MPL concessions.

Ophthalmophagocytosis confirmed the GMC results.

Materials and Methods / wfenfed / urt sd / drda

Different mothers come from. infant OFA rats selected randomly were immunized at 7 days of primary killing; additional booster vaccinations were given 14 and 28 days later. At day 56, blood was collected <2S days after vaccine 3). Each vaccine was administered subcutaneously, and 10 rats per os were administered.

The rats were immunized with an 11-wt pumiceococcal conjugate vaccine containing the following polysaccharide serotypes conjugated to protein Eb: 1, 3, 4, 5,68, 9F, 9V, 14C, 19F and 2F,

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The upregulation of the effect of the enhanced adjuvants on the production of larml is a constant dose of the conjugate taken from each poFstocharid, and. AlPÖ * and 3D-MPL adjm / ananthoh were prepared at various doses and corneal inclusions, including sgrgrant ileal case. 3 ,. • toMoz / mto APi'A rt?

Concentrated, adsorbent ntono valences are the; the following procedure; 58 [mu] g of AIPCcr {pB ~ 5.1> were mixed with 5 [mu] g of lubricated polysebaride for 2 hours. The pH was adjusted to 5, I. Leave the ancestor for another 16 hours. 1500mM NaCl was added to the mixture and a basalization of 150 ntM was performed after 5 minutes and 5mg / ml P-tonosieSanol was added to the mixture. With the passing of another 38 lawsuits, the pitch was set to ό, ί-rc, and left for more than four days in ʻC.

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Three solvents were prepared with 158 mM NaGilS mg / ml fesasietanoifean,

A: 1 mg / ml A1PO <

3: 3G-MPL in AlPoI, 258 and 1888 pg / ml, 5:28

C: 3D-MFL on AlPCh at 561 and 1888 pg / ml in SiO2, weight ratio 58:> 39

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Concentrated sporadic larvae FS-PD monovalent 1.1 were mixed in the correct shadow. You are given as AlP0 4 -actoglyceride as solvent A, where appropriate, as 3D-MPL or * as an aqueous solution adsorbed, 1 below), or as 3 or C gel solvent (3D-MPL AlPsA-ori in 2 doses, you will add 2. ot) below.

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To the combined adsorbed conjugates is added 3FA-MP1 as an aqueous solution. Up to 10 cycles of kcn-rocks and 1-vene edges were stored until administration.

3B-i ~ MPL. pre-adsorbed to AIPÖ <for the combined adsorbed conjugate. (solvent 3 and C). To prepare 1 mL of solvent, an aqueous suspension of 3D-MPL (250 or 561 µg) in 1 mg AlPO (4 µl) in 58 mM NaCl, pH 6.3 was stirred for 5 minutes at room temperature. This solution was diluted with NaCl pl, 1 / phenoxymethyl, and incubated overnight at 4 ° C.

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Eleven PS-PD conjugates were mixed in appropriate proportions and diluted with 150 snM NaCl, pH N / F. Where necessary, 3 D-MPL was added. solution (not adsorbed.

For all oysters: required blood pressure: 18 days before the first injection, we prepared an eb £ OSA

An ELISA procedure was performed to measure rat IgG (using the WBÖ Workshop Procedure for ELISA) to determine the serum levels of IgG antibodies to the anti-crepfococeas pnímwo ?? ine envelope polysaccharides. In essence, the radiolabel plate was directly coated with purified baro polysaccharide. The serum dips were pre-rinsed in all pt-eomococcal common cheese (pnslisacnariddda CC material) present in purified gneumococcal polysaccharides according to the disclosure of European Patent Publication EF 72513 Bt. Reagents from Jackson lineage stock exchange Ine. Were used to detect bound mouse IgG, The deletion curves were modeled using internal log-log equations for monoclonal antibodies, and the saturations were performed using Fo / Taphear / ke software. The maximum absolute snuff of these results is expected to be iron within a factor of 2. The relative error is less than 30%.

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The liters of opsostfers are determined for serotypes 3, S, 7F, 3.4, 1F, and 23F: the C3Gel procedure ("Siropenozoicoesa pneainooofee Ospsnnopbagoeytosis ttsiog lsrentisted HLoO", version: li) is used. strains, and the phagocytic I-lOO-cells are replaced with human neutroyl FMN (there is a high correlation between these two phage-sparing cells). In addition, 3 mm $ beads were added to increase microbial plate agitation, which allowed for a reduction in the phage phage to microplate ratio, which was recommended at 400.

Results for /^<í-,ío.vee written to>

The average geontact of the IgG chelating enzymes determined for each lpns and P1> rs is 4 ~ 100. TABLES HAVE BEEN PRESENTED, 14 ,. For the 19F and 23F serogroups, you will also get results with the previous stake formulation as a baseline,

The highest IgG machine impressions are highlighted in Figures 4-10. tables. The statistical checkbook is 313-MFL and 3D-M8LZAlPO. _t formulations are shown in Table 11. The high dose of absorbed conjugates with 313-MFL) gives the highest GMC value in 9 out of 11 cases and the low dose in 5 out of 13 cases). In addition, the use of sdjevant results in a higher GMC value than the dose adjustment of sserotype pouch (data not reported). and this statistically slips over the serotypes 4, 6B, 7F, IFC and 23F with radpsG.ÖS 95% ().

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The results of the collagen sera for openotagenesis are shown in Figures 3, 4B, 7F. i-L Í9F and 23F eyes! for apprentices, see Figures 4-8. In most cases, these opsonic titers support the GMC IgG. Indeed, the correlation with the IgG concentrate is greater, with 85% of the seronptts being ILS, IFF, and 23F (data reported). In the case of Srerotype 3, it should be agreed that only the Jö-MPL group elicited ophthalmic activity above the threshold.

findings

In this experiment, the only application of an unexpected blind, captured 3D-MPL is to blind the highest IoW.

By priming the adjuvant vacuole, the maximal GMF leG value is compared to the maximal CMC IgC value obtained by modifying the FS dose, and it is plated. The <RTI ID = 0.0> 3B-btPL </RTI> sssgnifiqm elicited a higher response with 1 i of 5 serotypes.

In Table 11, the 3-O-NÍPL and 3D-MPL / A1PO ₄ compositions összchasonlitásál Matata on (the removal process and the three O-MPL dose feasortiií good together), the poliszaebarid-Konjac rgátnmnk közül5 significant in favor of immunogeaitás respect., When it was formulated with only 3D-MFL4, instead of 3D-MPL and A1FO: FS 4, PS 68, PS 18C, FS 191 ”and FS 23F.

Example 3 - Fluorescence of the effect of the combination on the immunogenicity of PS 4, PS 68, FS 18, FS 19 F and FS 23 F conjugates in adult rats

Adult rats were chronically mimicked with pneumococcal polysaccharide-protin D conjugate vaccines or cd-dons, or in combination with muteival ready-to-use (ladder, penta, bepta, and deca). Groups of ten rats were knocked out twice at 28 days and blood was collected at age 28. On day 42 (14 days for the second dose dip),

Serum was tested with FLSA for IgO anti-pneumococcal anti-polysaccharides. Triggered by the production of specific IgG antibody from each of the canines based on the difference with EUSA, Table 12 shows the effect of combining saosov & feasible FS6B, FS18C, FS1FF and FS23P protein D &apos; s in their immunization clouds in rats. Based on IgG concentration 14 days after the second dose.

All samples were subjected to statistical analysis to determine that differences in costume biofeedback kiafakah were: Significantly, the combination of any of the PS6B, FS18G, PS19F and FS2SP protein B conjugates did not significantly alter their immunoglobulin concentration.

Table T: Specific Activation, Conjugation, and Reake bf-Disable KS r S toe s for FS-protem D Femjug Tour

Szerotlpos S 3 4 5 68 7F PS Cane, (Mg / ml) 2.0 3.0 2.0 7.5 5.4 3.0 FS solution 2M NaCl 2M NaCl MONTH I-i, 0 2M NaCl 2M NaCl PO Fenne, (Mg / ml) 5.0 5.0 5.0 5.0 5.0 5.0 Initial FS / FO ratio t> vrt *} 1/1 1/1 1/1 I / I 1 / t 1/1 COÁP Conc. (sng / mg FS) 0.75 0.73 0.75 0.75 0.75 9.75 pH _s / pH _k / pHj 9/9/9 0/0/9 9/9/9 9/9/9 9/9/9 9/9/9 Serotypic W 14 JSC 9F 23F

- 2 «Table 3: U-vshms pneumochexis PS-PB tested in infants

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H, Table; Statistical value (<p ~ value) that certain j taigigates imatuHbge & M & g get unique! 3Β »ΜΡί, -0« 8 packed in 3! 3spacious, (What do the G-s underneath a forest stgaitt,

Sero-types 50 μ «3D-MPL - SD-MPLÁAIPO ,, 5 hp 3D-MPL - 3D-MPX / A1PO ₄ 1, say 2, pass Road 1 2, pass 1 0.3 0.05 0,070 0.11 ³ 0,075 0.01 0.27 0,008 4: 0,002 0.0003 0.02 0,003 3 ^ 7 Ö.04 0,002 0.1 0.12 ............ »<ς · ....... 6B 0,001 0.0001 0,001 0.0006 7r 0.13 0.Í3 0.01 0,005 §V 0.02 0.02 0.1 0.04 74 0.65 0.21 0.3 Uh, 6o ! 8C .0808 0.0002 0,006 0,004 19F 0.0009 0,006 0.21 0.04 23P 0,002 0.0004 0.0 = 0.0004

2. Tabulate; Geometric Mean IgG Concentrators (pg / ml) for 14 days in the 2nd hnsswing of upper rats using 1.0 µg of polysodaryl-proline D supplement, individual desire combinatorial results, combinatorially, pentavalent, hepinavalent, or dexviable.

serotype 4 oB 18C 19F 23 F Vaccine H T 1-1 T ' T alone 9.3 8.11 15 5.2 2.5 combined 4 8.23 3.7 3.7 2.3 T: Combined with tetravalent ff) (FS 6S, 14, 19F, 23F), washable & Iens (T plus FS 3), heptavalent (Η) (7 plus FS 4, 9V and 1U), and decavatone (H plus FS 1, 5 and 7F) in the combined vaccine.14: Combined heptavalent: (Hj <T plus FS 4, 9 ¥ and 180, and: decavalent (H plus FS 1, 5 and 7F) in the combined vaccine.

<Example - Addition of Fnennmlizirt and 3B »MPL Snl to PO-lubricated IL-valent polysaccharide for beneficial effects against paenoxococcal gastrointestinal colitis

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Ap <RTIgt; rp </RTI> Nune mcrotter plates were plated for 2 h at 37 ^v G, 108 pg / vol 2 pg / ml in FBS diluent: Recombinant nativpsenmolysis (PEY), The plates were washed with 0.9% NaC. 0.05% Twees-20 Buffer, Then a series of two-fold dilutions of anti-PLY reference serum (PBS / 0.85% Twoen-20 Powder, added at 108 µl / well as standard curve (starting with µg / ml IgG) and serum fat; 1/1 G dilution) Incubate for 50 minutes at 28'Cma shaking After washing as described below, PBS / G, 05% Tween-20 diluted to 5800-fold in goat-conjugated goat perylidase anti-mouse IgG (Gackson) were incubated for 30 minutes at 2 ° C with shaking. After washing, the g plates were incubated for 15 minutes at room temperature. 188 thin wells (buffer: 8A gauge ODPA and 0.85% Η /.) In 108 tnM Euro buffer pH 4.5. Detection was also performed by addition of 50 µl / well of 1N I 4 Cl, and was read with 490 and λ20 nm under false pretreatment with Molecular Devices. The elienanysg titer was calculated using an O-parametric mathematical procedure using WtóAfer Pro software.

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This assay was performed to measure the ability of serum antibodies to inhibit the hemolytic activity of pnenmolysin (FLY), to remove cholesterol (susceptible to interactions with PLY), twice with the same volume of then incubated for 45 minutes on shaking. Supernatants were harvested on a 1008 rpm centrifugation tea for 10 minutes. The sera koleszterinmeates hlghottuk (double sorozathigltss dltietréltól 1 mM, 8.81% _BSA, 15 mM Tris, 150 NaCl, TNM, pHA, pofebes?) Main tsérőbdyss: jtókrotítórleírÍezen (bottom), 58 4 GB Otemollzb unit uk) FtY- A solution containing 4 was added to each well and incubated for 15 minutes at 37 ° C. Then 100 μ! sheep erythrocytes (1% solution) were added to the plates for 30 minutes at 3 ° C, l min, 1000 rptn estrliugaclofs, and their moles (150 pi) were collected and subjected to another §6 ~ : Leoing at 405 nm. Express the results in half-dilutions in twins.

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Recombinant native prismurnolism (PLY) was dialyzed against 5 .mu.M phosphate, 500 mM NaCl, pK-7.6. opposite. All subsequent steps were performed at 39.5'C with episodic stirring. On the first day, 10% Tween-SO (D'250 v.'v). 57.4 mM N-Acetyltriptyolane (H3O6 v-v) pH- 7.6. A mixture of 2.2 M glycine phosphate in poft (1/100 v / v) and 10% formaldehyde phosphate (3/100 v / v) in buffered form was added to the RLYel. On days 2 and 3, 10% formaldehyde solution was added again, incubation at 3/100 υ · ύ and 2/100 v / v in gold at A ~C for 7 days with occasional mixing. PLY is degraded with 50 mM NaCl, 5 mM, pH ~ 7, and is buffered in sacrificial holy. Total inactivation of PLY is

Seven-week-old female Oh f-mice: Intra-anaesthetically inoculated with 5x1 ff GFU mice into OB serotype with A. pssernowae. Lungs were removed 6 hours after infection and homogenized in (Ultramar, 24000 rpm, 4C) & W & (T148, Oibcos media). p &lt; tb &gt; &lt; / RTI &gt; lung infiltration f CFWeger, as logarithmic weighted mean.

Example 4 / Example WD / W £ heatwash.

Bobon a. In Example 1, the effect of the use of 3D-MFL adjuvant was evaluated on native recombinant pnoumoites (PL Vj J, Patou, Childrens Hosphnt, Norphal Awraka), and chemically detoxified (DPLYs) on the immune response.

Ten groups of six-week-old female ECU / C / C mice were sstmmaszkulfeisau inmtaMzáhank at 0 ,. 14 and 2L day 1 pg FEY acid v & w DFLY in PBS containing either A: 100 pg AlPO ₄ on _or S lo0 pg ÁlPCÁ plusz5 pg 3DMPL Í p-d-o-aeeliláit-monoítkszibrii lipid , Ribi Immtmoehem) Figures LA and Fig. 18 show the IgG and hemolysis inhibition titers of EL1SA after vaccination 3.

Either antigen, and, the best immune response was obtained in 3D immunized chickens immunized with 3D ~ M8t-lsi. An interesting modification of Í38LY was the same as the PLY, lm AlPÖ ,, / 3D ~ MPLs, and it has a tighter tin plume. With AlPfk Formulation, This has demonstrated the advantageous ability of 3D-MPL to enhance anti-detoxicap

In formulations containing pneumonol levels, it may be more advantageous to use chemically doxoxified pnearoolysin than mutant deoxygenates. The reason for this is that the dstoxified mutants obtained so far still have ridicidal toxin activity, but no chemically detoxified gmsmunization pathways. Thus, in general, another embodiment of the present invention is a descendant of a composition comprising a chemically-detectable imoumtshsm (or p-nenolysis), for use in a vaccine when co-administered with Ihl-sdjsvass, preferably 3D-MPI. A ~ 39 method for enhancing the immune response to chemically transfected poeamolysin antibody, comprising the addition of a Th 1 adjuvant (preferably 3D-MFL), is possible.

Example 4 / S éMsaonoFirst? ı.sy <ıugüc; í? ». v; dos and 3P-AÍPI mptóvdos Aosxá # <! ^ d« «V & * vol $ Arbbzu is protected by PD-Áöjj / Mgefc // '- va / ess pYjfAzozA'nróí-oirdunvog ? ACHIEVEMENTS OF FUCKING SEXY FUCKING FOUNDATION © / b «<zrón 'Ο / 7-egfíreá rWoáe / att & dáfcd & üK

In this example, a vaccine containing a propylene glycoprotein of l-valene polysaccharide-proline conjugate 13, attenuated mutant pumolysin strain (8dB, WO 90/06951), and adjuvant cause of A1P0 * and 3D-MPL was added. ssebssöaltfva a. classic .AtPCp adsorbate in 1 l-yskmx polysaccharide-prorem D-conjugate formulation.

Groups of 4-week-old female ÖEl-mice 12 groups were administered subcutaneously on days 0 and 14, with the following Kszerelssekhök A; 50 µg A1PÖ <; B: Ocular-conjugated polysaccharide oboe material containing 0.1 μη PS per wort; -t- 50 µg A1PO «; or C: D-conjugated polysaccharide vaccine containing 11 µg of serum, containing 0.1 µg PS, v 10 µε Papa (.1, Paton, Cilildren's 1-site, Nortb Athlete, Austin) + 50 µg AIPO <* 5 µg 3D- MPL (Emi ternnmochemj. Infection was performed on day 21 as described above).

As shown in Figure 1C: benurtattsk, 11-valent pfdiswlwid conjugate supplemented with PdB and .AS * O «* MPk-le gave significant (p <0.00?) Protection for the black rectangles shown by: arithmetic average). In contrast, no significant protection was found in animals immunized with the 11-pole PoksaBaride Tug Vaccine / AlFCE formulation. This result proved that the addition of the pnsumoltziu antigen (four of them attenuated) and the 3D-MPL adjuvant enhanced the efficacy of the 1i-valent polysaccharide conjugate vaccine against pneumonia.

Example 4 / C zt AS yébOóím femmaoíf shield immmm / ögm? mggA? kdöí

In Example 4B, the f1-valent polysaccharide conjugate with cottage, Icgv eugenic mutant pneumohistase sPdB) and 313-MPL: protection by immunoprecipitation, we measured pre-infectious, ßB polysaccharide and PB polysaccharide. antibody responses as described above.

Thereafter, the edible material titers were plated with the 6 h following the infection of the respective animals: the collected tttdejbeo measured hacteria-colony numbers. The R-values were calculated by logflog linear regression.

The calculated RNAiFcfe sequences were E, 18 and 0 ^ 02 for znti-PdB and attenuated antibody responses. This showed a lack of correlation between the Immoral tmtnutt response between the protective covers for both antigens :. The priced 1-68 slides were not significantly different in the groups immunized with the 1 L-conjugate vaccine (GMT - 0.3.18 sg?) Sl). or in the same vaccines supplemented with FdB and 3D ~ MPl. (GMT ~ 0.458 ng / ml?) Thus, the improvement in protection seen with formulation C is not solely due to the higher antibody response to 68-pollen.

Taken together, the results suggest that protection is not only mediated by the immoral immune response, but also by cell-depleted immunity induced by the presence of P6B allogene 3D-MPL. This provided additional support for the protein and antigen (s) and effective adjuvant (s) for the pneumococcal polysaccharide. , thus aligning both branches of jMMSta for optimal protection.

5. | febia ~ Co-fusion of both branches of the immune system is actively ionized in posutnolhsktne and psieantnkokksrsx ES dfeas ekcsaoyagokksi in passively hunsnnse mice

SZAsln ..... Finding passive hsadon sntÍ-BB-pohxzaeh & ri-sliensoyagag (anti-FS) lung inflammation protective machinery.

Vaccination groups: The mice group of mice are passively homogenized (mkageriosis). day, 100 μΐ dilution San rat anti-penetrant hydride anisle in the following esaporto. (total of 64 mice)

Aílulok; 64 brush CD - / - mice (Cfearles Riyer. Csnadn), weighing 3S g (surrounding gut).

Anesthesia: Mice (females (5%) plus O _; (1 L / min), pseudorasses: A. pncnisombé 'N1387 strain (S. Scrotipes) was harvested from 5% horse blood typhoid soyabean agar (TSA) dishes and 6 µl of PBS fu spe. Immediately before infection, 1 ml of bacterial suspension was diluted in 9 ml of cooled, melted nutrient medium and kept at 41 ° C. The mice receive approximately 10 doses of CFU mice in a volume of 50 µl.

Infection: On day 0, mice were challenged by the method described above and infected with strain S, _x feeW0> we N1387 (S0 pl by chilled fectenium-sspenovab intra-chronical drip, no surgical fetal rheumatoid arthritis). (Antunicrvh Ag. Ghemolkerap. 43, p. 29, 1999).

Samples: On day 3 of infection, 8 mice per group were sacrificed with G0> g, and lungs were excised and hbmogeized in 1 ml PBS, 10-fold serialized in EBS to determine the number of viable HCV. Samples were inoculated with (20 µl) 5% horsepower supplements in TEA and emergency, and 37 ° C through nightcap prior to evaluation. A further group of mice was sacrificed on day 7 and the nhbias were evaluated on the painting moth.

Scriven-Rred

IgC concentration (µg / ml) in rat serum |

Bacterial count (hanging from CFUMdo) after infection

6.7 * 0.7 (1/7), 27 0.7 (5/8)

6.9; 1.8 (4/7) 4.8 7 1.4 (2/8) C3 a T * 5 (3? 9> "

6.5 * 9.7 (1/7)

RJZoR'W)

1 $ ÁteÖ (3 / g)

The numbers in the zebrafish represent the number of animals that died before the time of sampling.

Following by:

Generally, there was no signi fi cant difference in the number of tuberculosis isolated from any of the treatment groups. This suggests that anti-polysiladd did not provide measurable protection up to and including 5 pg / ml.

It feels similar to what some human clinical studies have done, namely: atolliglation barrier for inadequate protection against pneumococcal pneumonia in certain populations,

Example S / B - Determination of Protection by Active Administration of PEY (Pneamobzin) with and without Adjuvant, and Synergy with Synoptic Ato-PS Matter,

BÜÉlb

Animals: 128 weeks? CD / mouse (Charles Kiver. St, Constant, Quebec, Canana at 6 weeks; Irish Immunization, It) at 7 weeks of infection. Animals weighed about 20 g at week 7 and about 38 g at 10 weeks. Immunization: Six groups of mice were immunized by subcutaneous injection of sub-tbt. and -14 days with 198 µl of a vehicle as follows (total 128 mice). Pd6 (WO 90.4) 6951. International Publication No. I) was obtained from the courtesy of Dr X, Paul, Atsstmia, SD-MPL from Kíbi / Corixa,

Specific on day-1: Esopsms (see box below) were passively immunized (109 µl islet batch) with 4.26 pg / l of mouse anti-polysaccharide antibody: (4 µl of 3 pg / ml solution κ 1.3 ml 2 pg) ml solution);

J-4 | -5 100 pl, Se. | MPL / AiFOsió'fÖ · knock ip a-PS good 100 μϊ, se. MPt / A! PÖ <(5-'50}

Infection; AU, day zz mice were anesthetized (with 3% isoll uranium plus 1 L / min Oj, Bacterial mucilypes A ^ mswamae strain NI387 saline serotype) were harvested from 3% horse blood trypticase soy agar (ISA) dishes. , 6 mL suspended in PBS. Ten-fold dilutions were made (plus 9 mL) of cooled, molten food steaks 1.4; C) immediately prior to infection The mice are infected with non-surgical thyrotracbic ductal tubing and received approximately ⁶ CFU / mouse of volume of 5 µg / volume. This procedure was described by Wöonut and Berry t Antimicrob Ag. 43, 29, (1999)); up

Samples; The infection was sacrificed at 72 hours per group by gavage with CO 2 and the lungs were excised and homogenized in 1 ml PBS. Ten-fold shuffling was performed in PBS to determine the number of viable bacteria. The samples were supplemented in 5 replicates in triplicate (20 µl). They were put on TSA dishes and hcubated at 37 ° C overnight: before evaluation. A further group of mice were sacrificed on day A and samples were evaluated as usual.

The ^ íoleteínsése

The number of bacteria in the comparator treatment was the number of bacteria in the lungs 3 and 7 days after infection. Results were expressed as the mean of the groups by standard deviation. Statistical analysis was performed by the Skiadele test, where a F value of .8 _and δ was maintained at a significant value.

It is our left-footed pity; In the i-4 group, they were significantly (p <S, 05) lower, in the matte group 14).

A: bacterial counts of esoporibates 1-4 were significantly (p <fkÜS) lower than those of 1-5

FUTURE -ibsb d a t & txés ortte

The Baltic mn-zan.s in each group were fatigued by two orders of magnitude lower &lt; tb &gt; day 3, showing eradication of infection.

Bacterial pacemakers significantly reduce I »2. (p «ö, ö5) were lower, nttef in i -5 groups.

Group J; 3 days 8th day j hanging CF'U / lung scatter hanging CELí / tUdö 1 Scatter Ϊ} 1-1 1 $ 93 ml 5.23 1 1.28 1-2 6:59 1.25 4.08t 1.34 | «1 7.09 0.8 5.32 ;, 2 O i-4 j 6.09 1.43 § 4.46 2.32 1-5; 7.19 0.89 5.42 ΐ 1:05 w j δ, 68 1,1,4 ⁵ 5.01: L. 1.48

Like their demosSSs, the Qoedalase autl-polysaharid counterpart (l-S) does not provide protection against the growth of pnenutococci in the stomach. FdS and AlPO ^ adjuvas also do not provide protection, but h. days, there is a trend toward protection if PdB is 3D-MPL-depicted (group 1-2).

A, 3 days; the most stable protecting group, Groups 1-4, all three components were Wp, PdS-1, 3-M'BL-1 and passively administered anti-polysaccharide antibody. This conclusion is supported by the mortality data. In Groups 1-4, 2/8 clears the wilderness with 5 to 0 deaths in Groups 1-5 and 1-3.

Ok Meal:

Further, the experiment was performed on passively immunized animals. to pnenmol izindcl and M'PL! The synergistic effect of active immunization with Pest was the result of an increase in the level of polysaccharide antigen antibodies.

Because the animals were only mimicking the passage of polish & eharid pueunsococci, by day 8 such antibodies largely disappeared from the vsh & host.

Even so, pneumolysin v 3D «MPL» lake is stagnant vs dendable-pneumococcal pneumonia; mmrumizate groups, and in particular, parenteral * 3D-MPL-leI -t-passively administered anti-polysobartd-sllsnspyagg in inosmized groups, showing the synergistic watering of this combination.

j) the adipyrite polysaccharide Ituploading was carried out actively (preferably conjugated polysaccharide). the effect yet; kitbjezotiehb would have been added since the effect of B cell memory and constant levels of Tto-PS antibodies were added to the immune response (see, for example, IC, where many horseradishes were detectable in horseradish and protein actively immunized with protein). ).

My protectors against ptemococcal strain infection are seipus-speel-specific antibodies. mediated by ophthalmophageal leesis * It is hypothesized that an increase in antibody concentra- tion is more protective of etiquette and thus devoted a great deal of energy to finding ways to enhance the honmrah's immune response. One of the strategies successfully employed in the pre-testing of kiloquits in conjugated vaccines is the use of mttnutsylmulsive adjuvants [Summary: Poolman et al., Carbobydrate-Bssed - 4 4 ~

The Baccotesian Vaccittesfe 'Líouöbook of Espmmmnmi Pbannaeologyfe edited by Perimans and. Wlgseii, published by Springnr-Veriag. Heiddberg, Ö (1998)).

Data revealed in this section: & Lego, Clinic! The results of an experiment using samples are presented in a clinic: a procedure designed to imitate exams.

procedure:

One-year-old 5 µg / µ2 mouse was immunized with the pneumococcal polysaccharide protein. The D-conjugate vaccine was administered at a dose of 1 / l human or 23-valent simple polysaccharide. The vaccines used were clinical specimens of DSPOöfe DSP013 or DSPO54, respectively, for the 1m dose of the conjugated vaccine alpha B and 23P serumHpus and the 0.1mg conjugate for the other i.v. . or 0.1 µg of dose of the 11-wafer vaccinated vaccine was 5 µM 3D-E. All 11-valnus conjugate vaccines were also formulated with SO pg ATO> adjuvant,

Groups of mice 20 were uninjured mtranmszknSarally. Vaccination of the groups listed in the table below was completed on days 8 and 21. On your 35th day (14 days after the second dose) a test bleeding was performed.

Table: Immunization of Sgyévss Rs / W mice! blueprint for pneumocephalus polylseharide prime ecjugate vaccine in clinical specimen!

Group yeah, day Vaccine Dose 1 Day 21 Vaccine Dose 2 Mice intended 1 Pneutnovax-23 2.5 meg Powder 28 2a 11-Pn Pn-PD up Buffer 20 2b 1 ί-waffle Pn-PD up 11-Pn Pn-PD 0.1 tneg 20 3a 11-Pn Pn-PD -r MPL 0.1 mege 5 meg Putfer 28 3b 11-Step Pu-PD - · MPL 0.1 meg s to 5 meg 1 t-valens Pn-PD ·; · MPL 0.1 meg ·? 5 meg 20 4a 11-Pn Pn-PD 1 / 8.5 meg Buffer 28 4b i 1-vak-ns Pn-PD 1 / 0.5 meg i1-pure Pn-PD i / 0.5 meg 20 control Pnffer ......... ............................. 28

Have you tested jvneumoccus using the BUREAU procedure? polysebsfids against the presence of IgA antibodies followed by CDC / WHO consensus ehanbn, ie serum; neutralization of the cell wall-walled stoneware. Calibration with EOSA mice lipid-friendly IgGi mosquito-like antibodies yields a concentration of mcg / ml in the presence of g / ml.

The statistical analysis of Oasebaspslhasas is computed using tWáTAT wsto 5.0 bem. ANÖVA scans were performed using the & gM.RTM. Method at most trans-ion concentrations. Pairwise comparisons of serotype-to-veridia ratios were performed using a Hisherelie exact test.

Bredmények:

The GMC fgG and serum 9559 n spiked against serotype 11 and protein D 14 days post-immunization are shown in the table below. Where 955 confidence mervaihwd you can not count a. smknnwzias proportional motel.

Group 1 shows the. For example, the effect of degeneration, which induces only IgM production in animals®. Most IgG concentrations below the detection threshold'w, however, the / te / h / Oegers were able to produce IgG · against some pneumococcal polysaccharides, namely &lt; 3, 19F and: 14.

The high serum-venation rate of hmnmnization with our conjugated fuel causes you to induce IgG-antiprotein production against all χ / erotypes except 23F,

IMzls-fhggé answers (2nd to 4th group) shares a. ? 'f and 19F serotype shocks were observed, but these were not statistically significant. Increased response was found after two doses (b groups versus a groups) for & 3, bB,? F and W assemblies and protein 13, and these were slatifically significant in many of the three formulations,

A. most interesting is the effect of 3D-MPL · The requested dose (group 3b) of the 313-MPL vaccine elicits the highest GMC of the specific IgG, and this is statistically significant for all sets except 23F, which is significantly higher seroconversion rate (p 0.02, group 3b versus group 2b, Fisher's exact test),

Table: Selecting the geometric mean of IgG concentrates of pBeotococcal serum sera and protein D and 95% corsphedenia of mice in one year Pu / b / C mouse

Group í 2a 2b 3 a 3h 4a 4b serotype GMíigöj ugmí (9598 KI) i GMjlgGj kg / ml (95% Κ.Γ; GMflgGl pg / ml (95% OFF) ÜMjlgGj pg / «ti (95% Ki) GMpgG] pg Oni <95% OFF) GM (IgG, µg / ml (95% OFF) GM (IgG) pg / ml (95% OFF) 3 0.24 (0.16-0.0) | 0.18 (0.11 to 0.27) (5.84 (0.47 to 1.5) 0.72 (0.51 to 1.0) 4.84 (3.0 to 7.9) 0.22 (0.14 to 0.35) 0.95 (0.19 to 1.8) .....SHE.......... 0.02 She 0:04 8/19 # 0.19 (0.09 to 0.41) 0.14 (0.07 to 0.27) 0.74 ¢ 0.29-1,91 0.09 (0.05-0.16) 0.11 (0.05 to 0.23) ? F 0.04 0 (# 20 0.07 (0.04 to 0.12) 0:19 (0.10 to 0.39) 0.15 (0.10 to 0.22) 0.97 (0.49 to 2.0) 0.09 (0.06 to 0.14) 0.45 (0.20 to 1.02) 14 0.15 3/20 # 4.5 (2.5 to 8.1) 6.2 (3.6 to 10.5) 12.9 : (7.8-21.2) 13.6 (9.4 to 19.7) 4.0 12,0-8.0) 6.9 (4.6 to 10.6): 19F 1.2 6.7 12.1 10.1 58.5 5.9 22.0

(0.56 to 2.6) (3.6 to 12.5) (7.6 to 19.3) (5.5 to 13.5) (42-01) (3.5 to 9.9) (16.0 to 30.2) 23F 0.07 Uh, OS Uh, O8 0.07 0.17 0.06 0.10 i COS 3/20 » 2/194 2/10 » u 2b ~ 1.13 ~ 4/204 PD * 0.25 5.2 11.9 13.5 93.0 10.0 33.7 1 / 20.4 (3J-b, 3i (6.9 to 20.7) (9.5 to 19.0) <49.1 to 195 · ¢ 0.4 to 18.4) (21.3 to 70/3):

* In the EUZmi unit; # smoconversion rate, twice standard deviation over mean negative control See table above for definition of groups.

The data disclosed herein should diminish that the addition of 3D-MPL to the liVate pneumococcal: polyhaloehyde protein D conjugate vaccine enhanced the immune response in all aged Se / W mice tested.

Most drops, two doses of vaccine triggered a higher geo-immunoconjugate concentration at a single dose; has also been observed in mammalian plaque polyaseeharrhid females, and is thus a sign of T-cell Dependent immune response and immune-evoked memory.

The data support a regimen for cough administration of a Tbl-adiovsoss (preferably 3D-MPI, -el) cough pneumococcal polysehydride vaccine, wherein at least two doses of the adjuvanted vaccine are administered over a period of 1 to 12 weeks. Scheme A is a further embodiment of the invention.

The mice used in the experiment are insensitive to PS 23 (plain or lambog). Interestingly, although elleosayagic kweweotransversion to pseudischaride remains low, depending on the vaccine formulation used, many more mice responded to PS23 when using 3D ~ MPL (adjuvanted significantly). The use of a Th1 adjuvant, preferably 3D-MPL, to alleviate the inferiority of the vaccinated individual in the pemphemococcal bovine barrier barrier is another embodiment of the present invention.

Example 7 - Alemer / mmriasgOkI G-pnshuehurid Protem D-Conjugate (PSG-PD;

THE; PROTEIN »EXPRESSION

As an example in Example 1,

B: MANUFACTURE OF C-POLI SAARIUM

The source of the C group of the polysaccharide is Λ '. umm; .gííí _t / f, v € 11 strain. This is fermented by classical fermentation methods (EP 625133). The dry powdered polysaccharides used in the conjugation process are identified with A-fenrergine-v (SS-Salugicals sa,).

An aliquot of the strain C111 was blotted and the slurry in 0.1 ml of yeast was 110% with dlalizate. v vi supplemented with: \ fa: Spread on a petri dish containing? ier Amze «medium, and 23-25 hrs of rabbits with 36'Con of Veggose (made in incubator,

The culture of icRzmt * contained thousands of npv resuspended pellets and was incubated with yeast / xA, supplemented with train dialysate (10% v / v), medium <RTIgt; edéeyí. The Mmr dish is scrambled for 23 to 25 hours by Se-C

- 4 <

incubation with water vapor in the inoculum _: the surface was again resuspended in Wetöen sterile medium with medium fetentation, and 0.2 to 0.3 ml of zen suspension was transferred to 12 other media.

After inoculation into a water-saturated Incubator® for 23-25 hours at 3 ° C, the culture was resuspended for one hour in 10 ml of sterile fermentation broth, from the bacterial suspension in a supernatant flask.

This suspension was then aseptically transferred using fertile sterile syringes.

The supernatant of rnemugokokhas was carried out in non-pressurized bathrooms; The férmentáoió corresponds to the total ehehével taUbaa 10-12 hours, to about 10 ^Io haktéríum / ntl's (i.e. the early stationary phase). and detected by an increase in pH.

In this condition, the whole medium is heat inactivated (12 eggs at 56 ° C) prior to centrifugation. Before and after Inactivation, we take from the culture medium and A / aferfer? spread on a petri dish.

C; PS-SCREENING

Purification is a multi-step process performed throughout the fermentation broth. In the first phase of purification, the inactive culture is clarified by centrifugation and the supernatant is harvested,

The polysaccharide purification is based on precipitation with a quinferner asninose salt (ethyl chlorinib dibromo mummum-bromtd, GTA-Cetvalon R). CTAB forms insoluble complexes poliamonokka ;, such _{poliszácbaridokkal,:} jmkleisssvaláiaf and proteins, p ^CODE:, depending ol. Following the ionically controlled reaction, this process employs the elimination of either fouling impurities (low conductivity) or pelisic debris (itself);

The clarified semisynthetic han _: insoluble phthalacahlates are triturated as a carrier (CeTlis * 545) as a carrier, in order to prevent clinging of the insoluble inert material during the precipitation step.

THE'. .Offer TriftridA 'C-peAswóötriiSffesaÁ · iozíMsi

Step L: A wedge of PSC-CTAB complex is fixed on Cellte® 545 and the cellular debris and proteins are removed by washing with 0.05% CTAB,

Step 2: PS is eluted with 50% ethanol. The first turbid fractions containing impurities and LSP are discarded. In the following fractions, the presence of PS is checked by a Phoenician test,

Step 3: PSC-C'fAB complexes are re-immobilized on Target® 545 and removed smaller ppklemic acids and proteins by washing with 0.05% CTAB,

Step 4: PS eluted with 50% ethanol. The first turbid fractions were discarded. For the following fractions, the presence of PS is checked by the Boccation test,

The eluate is filtered and the filtrate containing the crude PBS is collected. The dust carbide is precipitated from its hairs by adding ethanol to a final concentration of S0%. The polysaccharidof is then recovered as a white powder, dried in vacuo and passed at -20 ° C.

Bt CBAP CONJUGATION. «'C & Pö ÁOT / xfgí & main

For the conjugation of PSC and PD, the CDAP conjugation method was preferred over the classical CNIIar activation and coupling to the protein via a linker molecule. First the cis-vision of polishehandof! anyone who has l-cyano-4-disil: laminopyridine has an effect on fluorescence.

- <18 - (CDAP); CDAP is a water-soluble elastylating reagent in which it has an increased electrolysis rate compared to CNBr, allowing the crosslinking reaction to take place under any reasonably mild conditions. Upon activation, the pnllsuebridium can be directly coupled to the hydrogenose via its amino group without the use of any clamps, without the use of Vampfecal, mg reacting with and thoroughly reacting the AEROSPORES with glick to reduce the number of steps, no kapsfo-snslckns are present in the final product.

Activation of the polysaccharide by CDAF builds the cialis sperm and releases the mouse bacterium and dimethylaminopyridine (DMAP). The cyanate group reacts with mmsö-esopurts of the protein in the post-activation reaction and is converted to feadsamm tubule. F.WWW

Activation and switching is carried out at 2.5 ° C.

120 mg FS-tsldtmk for at least 4 hours in WF1 1 water for injection. Water fef irtjeeflcm '),

A solution of CDAP (100 mg in freshly prepared acetonitrile) was added to the polysaccharide solution until a ratio of 0.75 CDAWFS (WsV) was reached.

at 30 seconds, pi-taz activation! pH to <pH ~ 1) by the addition of triethylamine and stabilized until the addition of PD.

minutes to 30 seconds for MaCl sdrnrk to the mixture: In a 2M final concentre, apron purified FD is added to the mixture as 1.5 / 1 PD / FS; the pH is immediately adjusted to the coupling p1 (pK- ^: 10). The solution is allowed to stand for 1 hour under pH control.

, -4mM MM * ml of 2M glycine solution was added to the FS / Pf6 / GDAP mixture, pH is stopped! adjust to pH (pH ~ 8, δ); Stir at room temperature for 30 minutes, then overnight with 2-iFC stirring.

/ W FBfApírtfeu

After filtration (5 WW, the PD-PS co-jugate was cold-purified by gel filtration chro- nathrograph on Sbr / ueno, at $ 7s% W3?) For removal of cleavage molecules (including DMAP) and co-conjugated PD, Helel, pH 6; , 5; follow-up; UV2 &lt; nm, pH and conductivity.

According to the different molecular sizes of the reaction components, the PS-FD conjugate is eluted first, then free PD and finally DMAF. Fractions containing conjugates detected with DMAB-bn1 (PS) and pBCA (protein) were pooled, Az; the pooled fractions were sterile filtered (0.2 µm).

E; REMOVAL OF ABSORBED PSC-PÖ-KÖJJEJE VACUUM

AfPíC AUUVOS

To optimize the adsorption of FSC-FD-conjugate AIPO <, wash AIB <M to reduce the PO./'- concentration:

- AlPO ₄ was washed with 150 cc-1 HaCL and centrifuged (four times;

the precipitate was resuspended in 150 µl NaCl and filtered (18 µm); and

- sterilize with the supernatant, known as washed AiPCfet W'AP ("washed autocksved phosphate").

K & zere / uní eri4 «r .->

A large amount of PSC-PD conjugate was adsorbed on AlPO $ WAP prior to formulation of the ts final product. A1PO, WAP was mixed with ESC-PD for 5 minutes at room temperature. The .pH was adjusted to SJ-rs and the mixture was stirred for an additional IS hour at room temperature. Adding hlaCi solution to the mixture was mixed with 1.50 tMM of corsica and ssubbed for 5 min. d-Phenoxyethyl was added at 5 mg / ml and the mixture was stirred at 15 pervtg for 30 minutes and then reduced to reflux.

kegsó ariaarei irriiririr - ESC-ED: horse pgPS - AIPO, _Wai>: 0.25 mg? -KaCh ISttmM - 2 x © xietanoí: 2.5 mg - water for injection: To 0.5 ml - theirs ó.l

FOREIGN PRINCIPAL FACILITIES

FoilSrtnív5sívd-, lfjuíi <gdte> s fet, »; <r« i? G;? Í?; 7dro mouse & e »

The flesh of itnmmsogen in the PSC-PD score jug was determined in fo8-week old Zfo / h / C mice. The conjugate with plain (unabsorbed) ointment or absorbed on AIPO <is monovalent: the vaccine is Infected, and the induced anri-ESC material is eluted with ELISA. while running chemisexuals were analyzed for hachteridia, both methods were CXX? (Centere for Dtsease Contre), by Eeven / ion, Atlanta, VSA, I present the results of two different experiments to determine the effect of dose and adfuvant t'AIEOC,

Ddris-áfeis áriórií ?? '

In this experiment, the PSC-EO conjugate was injected twice into E1 rriCC alarms, with two font sizes. Four different AlPfri-rm conjugated dose formulations were used: 0.1; 0.5; 2.5 and 9.5 µg / animal. The mice (group Suff) were challenged at 14, (14 days after the first vaccination), 28 (14 days after the second vaccination), and 42 (28 days after the second vaccination). Geometric portions of C-polysaccharide-specific antibodies were expressed as tOMC) ug igG ml, using the graying IgG reference. The bacterial antibodies were dipped in compounding sera and the liters were halved to 50% of the horseradish diluents as a diluent for diluent, strain Λ ¹ , wefesgiridri Cl 1: alkabhazya nvah loseesemö complete in the presence of? -ο§ starting with the dose. The results indicate that there is a good booster between the 14th day after the first vaccination and the 14th day after the second vaccination, 28 days after the second vaccination, the antibody levels are at least equivalent to the 14 days after the second vaccine mum. The bactericidal nutrient shells are in agreement with <RTI ID = 0.0> B, </RTI> iSA-derived cortex and confirm the immunity to PSC-FB.

In this; in the experiments, one miot of our PSC-PD conjugate on AlPO was conjugated with 3 smooth non-adjuvanted injections. 19 mice per group were injected. at intervals of two weeks, subcutaneously after administration with 2 g of kommunis. Mice were bled at 14 (14 days after the first vaccination), 28 (14 days after the second vaccination, and 42 (28 days after the second vaccination)) and tested for tears in EUSA and functional etlenanagents. 14 days after the second vaccination and 28 days the second cause, for the Bacterial Test, the AlPfk formulation exhibits up to 5 times higher antibody stimuli compared with anemone adjuvant tests,

AlpveífeWírtseá

Stable general conclusions can be drawn from the results presented above;

The PSC-FD conjugate elicits a memory response demonstrating that · the conjugated PSC becomes a T cellDlggd antigen,

Antil-PSC concentration (measured by ELISA) is well correlated with the bskiericide antibody titres, indicating that the PSC-PB conjugate-eluted rabbits are functional a; Against its serogroup C (?)

- Approximately 2.5 (tg of adjuvant adjuvant on AMA appears to elicit an optimal immune response in mice; ·.

The CDAF reaction appears to be a process for the preparation of a smmmogenic PSC-PD-block inhibitor.

Example 8 - Polymer of A.Wamgri from A-Separation Group Elimination of Blonjug-Prnfetn Blionjugain;

Polysaccharld-A (PSA) dry powder was dissolved for one hour in 8.2 M NaOH solution for 8 ms / ml final kone extraction. & PB was then set to 6 with either HCl or BaQB and the side temperature was controlled at 23'C. 9.75 mg CDAP / mg PSA (109 mg / ml in acetone / l) was added to the FSA solution. After 1.5 oves (pi control), 0.2 M NsOK was added and a pH of 10 eS was obtained, and protein D (concentrated to S tng / tnl) was added 2.5 minutes later. , approximately 1 PD / PSA ratio. The pH of 18 was maintained during the coupling time of 1 hour. Then, 18 mg of glycine (2M, pB-- ~ 9.0) / mg FSA was added and the pH was adjusted to 9.0, 3 (for 1 min at 25C). The mixture was then kept at 4C overnight before purification by gel &amp; O chromatography (Sepnotv ^ from PhortRaeia-lblb) The conjugate was eluted first, then unreacted PD and byproducts (BMAP. glycine, salts). The conjugate was collected and sterilized by filtration at 0.2 n-methanol (Sar-orms).

Example 9 - Ν 7 and%, mW signaling of pAWnm efoWfed fermdfefe guard

The main features are summarized in the table below:

Woman. description of kon ingáin® Protein and PS content spgAtil) PS / protein ratio fiv / w) Free protein (%) Free PS : 1% 1 PSC-PD NaOl or sab. pH PD: 210 PS: 308 1AW <2 9 g 2 PSC-PD TEA va! tailor. pH. PD: 230 PS: 351 1 / 0.65 <2 5-b 3 PSA-PD NaOH, pH FD: 159 PS: 149 1 / 1.07 j 5 5-9

/ ?? son-··, ::? medium metal mice / Z / C mice were used as animal models to investigate the immunogenicity of conjugated metals. The conjugates were either adsorbed to A! PO _r rc or Al (OH> _$ (10 pg PS to 500 pg Af'), or Ter was adsorbed, A? weekly intervals (2 pg PS / vaccine).

To these results! first, we conclude that free PS greatly suppresses the immune response. Better pine needles: we got it! less FSA with wood-based cognate gums The above improvements to sn CnAP are thus a further aspect of the invention.

The presentation is also important. AlPO, seems to be the most adjvantant ehbest in the model. The mantle glands exert a reinforcing effect, and a series of single-injected polysaccharides, heads, and gums are observed.

The Ai memAgifluoride A and C polysubbubes were produced with an average protein ratio of 1 and δ, 6-P, 7 (va'w) ps · '. Free PS and free borosafe were IWs and 15% aphid, respectively. The yield of poysachan was higher than 70%. The FSA and PC conjugates obtained by the above improved ÜDAP screening are ligated to ribbed proteins (preferably with protein D), thus forming a further embodiment of the invention, e.g. the production of pofiskaehafed-profem fi-krmj egal fi. Mr W «« Mr is one of the major causes of meningitis in children under the age of two, the Loophole Polys conjugate of the Influenza Influenza (PRP) is well known. Reaction developed by Robbins! komugalva). Stats for CÖAP is an improved rape salt. Description of the optimal pathogen found when conjugating s PRP (preferably for Pi).

The parameters that influence the conjugation: reaction are as follows:

* Polysaccharide Release Concentration (which may have a dual effect on the final level of the free polysaccharide and the sterile filtration step), »Initial Concentrator of the carrier protein, , * The amount of CDAP used is usually used in a large excess), * The heat of application! (which may influence the degradation of the polysaccharide, the kinetics of the reaction, and the isoflow of the reactive groups), * The activation and coupling pi, * The pH of the reaction stopping (affects the level of residual DMAP).

* Activation, Switching and Shutdown Time: We have found that three of the most critical parameters for optimizing end product performance; · initial protein ratio; polysaccharld source coscene &cache; and the pH of the coupling,

Therefore, we design a 3-parameter reaction system with the above three conditions at the three-weak level. The focal points (and experimental ranges) were ?, FS / protein ratio; 1/1 (? 0.3 / 1); [?] 5 mg-'ml (± 2 tng'm?}; Switching pH; 8.0 (.s 1.0).

The less material; parameters were fixed as follows: 30 mg petroleumephearidol; temperature 25 ° C; (CDAP1 ~ 8.75 mg / mg AND; pH-0 (adjusted with 2M biaH); activation pH 9y5; activation time 1.5 minutes; switching time 1 hour; [protein - 10 mg / ml; stop pH 0.0; Stopping time 1 hour; PS dissolution time in solvent: 1 hour in 2 M NaCl; purification on a Seponneer / S4? / A column with ISO mM NaCl at a flow rate of 12 em / hour and sterile filtration on a P2d filter at 5 ml / min.

The data tested to determine the optimized conditions for the larvae described above were: process data - maximum yield; maximum level of protein incorporated after filtration; and burial quality ;. data - final FS / protein ratio, free AND level, free hinge color, minimum residual DMA.? (CDAP Degradation Product Synth

Your hair? Jio »

The factor influencing the post-fertilization rate after filtration is the interaction between khndaiúsr [Pb] and the switching pHl and the hardening AND / gland ratio. Low (ANDj omit milletSmsatss with the two latter factors and always have good yields (about 95% for all products), However high cooalmalt salts: the solubility decreases la the pH and the starting ratio ts (core [AND], lowest ratio, lowest pH 99% filterability; but high 1FS], highest ratio and pH 19% filterability).

The final ratio of PS / protein relative to the starting ratio is the measure of coupling efficiency. At high [PSj, pH is not affected. ratios. However, the starting rate is yes (1.75 for a low starting rate, 1.26 for a high starting rate). At low [AND], the ratio would be much lower but pi-l has a higher cooling rate (low pH, low ratio 53

0.96; low pH, high ratio ~ 0.8; high pH, low ratio - 1.4 and high pH, high ratio ~ 0.02).

The final ratio depends on the starting ratio and FS FS. The highest final ratios are obtained with a high baseline and high [PS] cortisol. The effect of pH on the final ratio is not so significant. -like -low [FS j,, $ W <ith & lint

The smallest amount of free protein at high pH Dt and high (FSI & M Sliced »5og & level (;, (:), A high (FSj effect becomes particularly pronounced when pH is low, increasing baseline is a delay) increases the level of free protein: O.

Fe-faded SAH?

The baseline ratio has no significant effect. With this, DMAE levels increase with (FSj) in food and decrease when the pH is raised.

ÁFvuákezrerdsek

Thus, the more conjugating conditions of the respirator are as follows coupled to a pH of 9 - 9 - (FS) -? and a starting ratio of ~ 1 / l, under these conditions, the properties of the final product are as follows;

Final rate FS / protein FS yield after filtration (%) Ratio of ratios SzaMd protein 15 (%) DMAF level (ngí 'l pg pg) value province value province value province value province value province 1:10 0.91 to 1.30 92.6 50-138 1.16 1.03 to 1.29 0.71 0 to 10.46 4.95 2.60 to 7.80

Thus, FFF coagogues obtainable by CDAP (optimized} below, independently of the carrier protein, but preferably protein H) thus constitute a further embodiment of the invention.

Π, Example: Frotein 0 as Antigen - How to Improve the Non-Typical //, ö? / & Guw elüsd: Protective Effectiveness by Formulating ÁD-MPk

Female SEMEMA mice brush (IU per group) immigrate (imramuscular) to the H-valent Paleumococcal Polysacbsnd Protein D-conjugate, first in 2O doubtful (D0), then second two weeks later (1). Seven days after the second immunization, the blood was seeded, and the liters of protein S etlni were measured for the amount of IgG 1, IgG2a and IgG2b antibodies.

Fagv: Astra-dried H-valent vaccine (without AIPO ₄ ) was prepared by combining the conjugates with 15.75% lactose with stirring at room temperature for 15 minutes, adjusting the pH to 6.1 µl, and lyophilized (started at -69C at the start of the cycle, gradually resolved to -24C over 3 hours, then held this boom finding for 18 hours, then gradually reset to -16C, then maintained at this temperature for 6 hours, then gradually adjusted to 34 ° C over 3 hours, then maintained at 9 ° C).

The composition of the formulations for reconstitution and reconstitution is shown in Table 13.

The most characteristic assay for Tbl-cell-mediated Immune Response was a known correlation with the level of IgG2a antibodies. As can be seen from the data, a surprisingly large increase in IgG2a-knot co-transduction results from the fact that the protein Da W -aápnAssssl (in this case, 3 D-MPL -?

, sv '•', ν ·.

Claims (9)

PATENT REQUIREMENT f. inenuaegéo kit containing several pofea.el el arid4teoiogattsm antigens which bind to aa <??? söpSSíiX PgtoswzHe protein D protein or to a? head. 2. The t. The orinogenous preparation according to claim 1, which is a peptide of the invention, which is a polypeptide, a polysaccharide of B-esopenos, a polysaccharide, and a polysaccharide. ogoMoíloe poiiszaobsrióok, StzsgyeeoeeKs -peliszaehatridok, nbkebaktétintncík pobszaehetidísk, Cryproeeeoas nenpírnníns poiiszsotoiáok, typified .oeot AgznígphíZKr íítBaenana ílpopotiszáelraridek, Aosr Oyobi ?? 's ífpwozög b & zaeharldok loop-po, Aforaxs / chloro οοίΖοίτο / Α HpopohszaeMrfeok, so BétgaBo ??,? lipepoziszaeharidok Ife, or 7iyp «« o.vomama force i: any of the IPPPGs. The rounenogenic peptide protein according to claim 2, which authorizes at least four hyperthyroid pneumothoraxic polyethylene glycols. 4. The itungmogenic composition according to claim 2, wherein the protein feeblastic antigens are fused to β? WinonPtglrkB? Coinbiotic legs of A, C, or V sarlotslns are derived. The non-intrinsic Honeycomb of Claim 1, wherein the honeysuckle contains a polysocstylidyl polysocstidar of n-enfegococcus C and an iningngococcal iog, wherein our skin; ·· gossip bushes-poksa hariboniini baths bathed with Ai OyAonaara protein D proteins, with the proviso that / Αηοηορ / ΡΡοί / g / Benzoí? ρ burgk-bobbsarkar to fetan toxin -vax conjugates. The tin onogenic composition according to claim 1, which is Srephocoams. r? nns? ko;? íoe, a · rnsomopóíDs gyAiensoíí b. does the Oeeningococcal C and the Earth Agococcal Y contain the coagulated envelope of the envelope, where the boning is given to the btank-giohsaobarlp'-sni? protein. .0. proteins are cotagogic, with: the caveat, the tip of the BoemophibíS Oytíem Ö wines · 'pelsszaebsritbö tetant'szAoxoidbez vaxs coo A nuuviogenic composition according to any one of the preceding claims, which further comprises an adjuvant, A lutumogenic composition according to claim 7, wherein the polyazoarlo protein Xbkotsjagam. antigens are adsorbed to aluminum phosphates. The mmnam gene composition according to claim 7, which comprises a preferebral inoculum of a Th I response. ii) The sustained release composition according to claim ih, wherein the edema is at least one of the following: 3D-WL, Sggonin Immunostimulant or Immunorimulatory CpG 11, Preparation of Ingnonogen according to one of the preceding claims. as a medicament for breakdown use. Also the I-tb. Claims according to Claims 1 to 4 containing a cotyledonous Iotomnngene composition. 13, Procedure for Multiple Pathogenic Foal Space Ioanormgenic Preparations of Dehydrogenase,. isolating glandular bacterial glandular glandular steroids; for the salts of the persian, I choose ours; .peBsachands for the protein D protein. koitjngáljuk; and mixing the conjugated poly saccharides. Use of a cold amount of a nonthmogenic composition according to any one of the preceding claims for the manufacture of a medicament for the induction or initiation of infection in a bacterial p &lt; tb &gt;